Enzymes and methods for producing omega-3 fatty acids

ABSTRACT

The present invention relates generally to the field of recombinant fatty acid synthesis, particularly in transgenic plants. The application describes genes involved in fatty acid synthesis and provides methods and vectors for the manipulation of fatty acid composition of plant oils. In particular, the invention provides constructs for achieving the integration of multiple heterologous genes involved in fatty acid synthesis into the plant genome, such that the resulting plants produce altered levels of polyunsaturated fatty acids. Also described are methods for enhancing the expression of fatty acid biosynthesis enzymes by co-expressing a silencing suppressor within the plant storage organ.

This application is a continuation of U.S Ser. No. 13/129,940, filedSep. 30, 2011, now allowed, a § 371 national stage of PCT InternationalApplication No. PCT/AU2009/001488, filed Nov. 17, 2009, which claims thebenefit of U.S. Provisional Applications Nos. 61/270,710, filed Jul. 9,2009; and 61/199,669, filed Nov. 18, 2008, the content of all of whichare hereby incorporated by reference into the subject application.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“140715_2251_79998_AA_PCT_US_Sequence_Listing_AHC.txt,” which is 134kilobytes in size, and which was created Jul. 15, 2014 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Jul. 15, 2014 aspart of this application.

FIELD OF THE INVENTION

The present invention relates to methods of synthesizing long-chainpolyunsaturated fatty acids, especially eicosapentaenoic acid,docosapentaenoic acid and docosahexaenoic acid, in recombinant cellssuch as yeast or plant cells. Also provided are recombinant cells orplants which produce long-chain polyunsaturated fatty acids.Furthermore, the present invention relates to a group of new enzymeswhich possess desaturase or elongase activity that can be used inmethods of synthesizing long-chain polyunsaturated fatty acids. Inparticular, the present invention provides ω3 desaturases, Δ5 elongasesand Δ6 desaturases with novel activities. Also provided are methods andDNA constructs for transiently and/or stably transforming cells,particularly plant cells, with multiple genes.

BACKGROUND OF THE INVENTION

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFA and VLC-PUFA)are now widely recognized as important compounds for human and animalhealth. These fatty acids may be obtained from dietary sources or byconversion of linoleic (LA, 18:2ω6) or α-linolenic (ALA, 18:30ω3) fattyacids, both of which are regarded as essential fatty acids in the humandiet. While humans and many other vertebrate animals are able to convertLA or ALA, obtained from plant sources, to VLC-PUFA, they carry out thisconversion at a very low rate. Moreover, most modern societies haveimbalanced diets in which at least 90% of polyunsaturated fatty acids(PUFA) are of the ω6 fatty acids, instead of the 4:1 ratio or less forω6:ω3 fatty acids that is regarded as ideal (Trautwein, 2001). Theimmediate dietary source of VLC-PUFAs such as eicosapentaenoic acid(EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3) for humans ismostly from fish or fish oil. Health professionals have thereforerecommended the regular inclusion of fish containing significant levelsof VLC-PUFA into the human diet. Increasingly, fish-derived VLC-PUFAoils are being incorporated into food products and in infant formula,for example. However, due to a decline in global and national fisheries,alternative sources of these beneficial health-enhancing oils areneeded.

Higher plants, in contrast to animals, lack the capacity to synthesisepolyunsaturated fatty acids with chain lengths longer than 18 carbons.In particular, crop and horticultural plants along with otherangiosperms do not have the enzymes needed to synthesize the longerchain ω3 fatty acids such as EPA, docosapentaenoic acid (DPA, 22:5ω3)and DHA that are derived from ALA. An important goal in plantbiotechnology is therefore the engineering of crop plants which producesubstantial quantities of VLC-PUFA, thus providing an alternative sourceof these compounds.

VLC-PUFA Biosynthesis Pathways

Biosynthesis of VLC-PUFAs in organisms such as microalgae, mosses andfungi usually occurs as a series of oxygen-dependent desaturation andelongation reactions (FIG. 1). The most common pathway that produces EPAin these organisms includes a Δ6-desaturation, Δ6-elongation andΔ5-desaturation (termed the Δ6-desaturation pathway) whilst a lesscommon pathway uses a Δ9-elongation, Δ8-desaturation and Δ5-desaturation(termed the Δ9-desaturation pathway). These consecutive desaturation andelongation reactions can begin with either the ω6 fatty acid substrateLA, shown schematically as the upper left part of FIG. 1 (ω6) or the ω3substrate ALA, shown as the lower right part of FIG. 1 (ω3). If theinitial Δ6-desaturation is performed on the ω6 substrate LA, theVLC-PUFA product of the series of three enzymes will be the ω6 fattyacid ARA. VLC-PUFA synthesising organisms may convert ω6 fatty acids toω3 fatty acids using an ω3-desaturase, shown as the Δ17-desaturase stepin FIG. 1 for conversion of arachidonic acid (ARA, 20:4ω6) to EPA. Somemembers of the ω3-desaturase family can act on a variety of substratesranging from LA to ARA. Plant ω3-desaturases often specifically catalysethe Δ15-desaturation of LA to ALA, while fungal and yeast ω3-desaturasesmay be specific for the Δ17-desaturation of ARA to EPA (Pereira et al.,2004a; Zank et al., 2005). Some reports suggest that non-specificω3-desaturases may exist which can convert a wide variety of ω6substrates to their corresponding ω3 products (Zhang et al., 2007).Other ω3-desaturases may have a preference for ω3 substrates (Sayanovaet al., 2003).

The conversion of EPA to DHA in these organisms is relatively simple,and consists of a Δ5-elongation of EPA to produce DPA, followed by aΔ4-desaturation to produce DHA (FIG. 1). In contrast, mammals use theso-called “Sprecher” pathway which converts DPA to DHA by three separatereactions that are independent of a Δ4 desaturase (Sprecher et al.,1995).

The front-end desaturases generally found in plants, mosses, microalgae,and lower animals such as Caenorhabditis elegans predominantly acceptfatty acid substrates esterified to the sn-2 position of aphosphatidylcholine (PC) substrate. These desaturases are thereforeknown as acyl-PC, lipid-linked, front-end desaturases (Domergue et al.,2003). In contrast, higher animal front-end desaturases generally acceptacyl-CoA substrates where the fatty acid substrate is linked to CoArather than PC (Domergue et al., 2005).

Each PUFA and VLC-PUFA elongation reaction consists of four stepscatalysed by a multi-component protein complex: first, a condensationreaction results in the addition of a 2C unit from malonyl-CoA to thefatty acid, resulting in the formation of a β-ketoacyl intermediate.This is then reduced by NADPH, followed by a dehydration to yield anenoyl intermediate. This intermediate is finally reduced a second timeto produce the elongated fatty acid. It is generally thought that thecondensation step of these four reactions is substrate specific whilstthe other steps are not. In practice, this means that native plantelongation machinery is capable of elongating VLC-PUFA providing thatthe condensation enzyme (typically called an ‘elongase’) specific to theVLC-PUFA is introduced, although the efficiency of the native plantelongation machinery in elongating the non-native VLC-PUFA substratesmay be low. In 2007 the identification and characterisation of the yeastelongation cycle dehydratase was published (Denic and Weissman, 2007).

VLC-PUFA desaturation in plants, mosses and microalgae naturally occursto fatty acid substrates predominantly in the acyl-PC pool whilstelongation occurs to substrates in the acyl-CoA pool. Transfer of fattyacids from acyl-PC molecules to a CoA carrier is performed byphospholipases (PLAs) whilst the transfer of acyl-CoA fatty acids to aPC carrier is performed by lysophosphatidyl-choline acyltransferases(LPCATs) (FIG. 2) (Singh et al., 2005). The reduction in flux due to anacyl-exchange having to occur before desaturation can follow elongation,or vice-versa, may be overcome by using a desaturase that hasspecificity for acyl-CoA substrates (Hoffmann et al., 2008).

Engineered Production of VLC-PUFA

Most VLC-PUFA metabolic engineering has been performed using the aerobicΔ6-desaturation/elongation pathway. The biosynthesis of γ-linolenic acid(GLA, 18:3ω6) in tobacco was first reported in 1996 using aΔ6-desaturase from the cyanobacterium Synechocystis (Reddy and Thomas,1996). More recently, GLA has been produced in crop plants such assafflower (73% GLA; Knauf et al., 2006) and soybean (28% GLA; Sato etal., 2004). The production of VLC-PUFA such as EPA and DHA involves morecomplicated engineering due to the increased number of desaturation andelongation steps involved. EPA production in a land plant was firstreported by Qi et al. (2004) who introduced genes encoding a Δ9-elongasefrom Isochrysis galbana, a Δ8-desaturase from Euglena gracilis and aΔ5-desaturase from Mortierella alpina into Arabidopsis yielding up to 3%EPA. This work was followed by Abbadi et al. (2004) who reported theproduction of up to 0.8% EPA in flax seed using genes encoding aΔ6-desaturase and Δ6-elongase from Physcomitrella patens and aΔ5-desaturase from Phaeodactylum tricornutum.

The first report of DHA production, and to date the highest levels ofVLC-PUFA production reported, was in WO 04/017467 where the productionof 3% DHA in soybean embryos is described, but not seed, by introducinggenes encoding the Saprolegnia diclina Δ6-desaturase, Mortierella alpinaΔ6-desaturase, Mortierella alpina Δ5-desaturase, Saprolegnia diclinaΔ4-desaturase, Saprolegnia diclina Δ17-desaturase, Mortierella alpinaΔ6-elongase and Pavlova lutheri Δ5-elongase. The maximal EPA level inembryos also producing DHA was 19.6%, indicating that the efficiency ofconversion of EPA to DHA was poor (WO 2004/071467). This finding wassimilar to that published by Robert et al. (2005), where the flux fromEPA to DHA was low, with the production of 3% EPA and 0.5% DHA inArabidopsis using the Danio rerio Δ5/6-desaturase, the Caenorhabditiselegans Δ6-elongase, and the Pavlova salina Δ5-elongase andΔ4-desaturase. Also in 2005, Wu et al. published the production of 25%ARA, 15% EPA, and 1.5% DHA in Brassica juncea using the Pythiumirregulare Δ6-desaturase, a Thraustochytrid Δ5-desaturase, thePhyscomitrella patens Δ6-elongase, the Calendula officianalisΔ12-desaturase, a Thraustochytrid Δ5-elongase, the Phytophthorainfestans Δ17-desaturase, the Oncorhyncus mykiss VLC-PUFA elongase, aThraustochytrid Δ4-desaturase and a Thraustochytrid LPCAT (Wu et al.,2005).

There therefore remains a need for more efficient production of LC-PUFAin recombinant cells, in particular in seeds of oil-seed plants.

SUMMARY OF THE INVENTION

The present inventors have identified for the first time a Δ5 elongasewhich efficiently converts EPA to DPA in a recombinant cell.

Accordingly, the present invention further provides a recombinant cell,preferably a plant cell and more preferably a plant seed cell,comprising an exogenous polynucleotide encoding a fatty acid elongasewith Δ5 elongase activity, wherein the elongase has activity on EPA toproduce DPA with an efficiency of at least 60%, at least 65%, at least70% or at least 75% when the elongase is expressed from the exogenouspolynucleotide in the cell, preferably in a plant cell.

In one embodiment, the elongase comprises amino acids having a sequenceas provided in SEQ ID NO:6, a biologically active fragment thereof, oran amino acid sequence which is at least 47% identical to SEQ ID NO:6.

In another embodiment, the cell further comprises exogenouspolynucleotides encoding;

i) a Δ8 desaturase and/or a Δ6 desaturase,

ii) a Δ9 elongase and/or a Δ6 elongase,

iii) a Δ5 desaturase, and

iv) optionally a Δ4 desaturase and/or an ω3 desaturase,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell.

The present inventors have also identified an ω3 desaturase with novelproperties. The ω3 desaturase is useful in recombinant pathways designedto yield EPA, the downstream fatty acids DPA and DHA, and other ω3VLC-PUFA.

Accordingly, the present invention provides a recombinant cell,preferably a plant cell and more preferably a plant seed cell,comprising an exogenous polynucleotide encoding a fatty acid desaturasewith ω3 desaturase activity, wherein the desaturase is capable ofdesaturating at least one of ARA to EPA, DGLA to ETA, GLA to SDA, bothARA to EPA and DGLA to ETA, both ARA to EPA and GLA to SDA, or all threeof these when the desaturase is expressed from the exogenouspolynucleotide in the cell.

The desaturase is preferably a front-end desaturase.

In another embodiment, the desaturase has Δ17 desaturase activity on aC20 fatty acid which has at least three carbon-carbon double bonds inits acyl chain, preferably ARA.

In another embodiment, the desaturase has Δ15 desaturase activity on aC18 fatty acid which has three carbon-carbon double bonds in its acylchain, preferably GLA.

The desaturase preferably has greater activity on an acyl-CoA substratethan a corresponding acyl-PC substrate.

In one embodiment, the acyl-CoA substrate is ARA-CoA and the acyl-PCsubstrate comprises ARA at the sn-2 position of PC.

In yet another embodiment, the cell is a plant cell and the desaturasehas activity on ARA to produce EPA with an efficiency of at least 40%when expressed from the exogenous polynucleotide in the cell.

In one particular embodiment, the desaturase comprises amino acidshaving a sequence as provided in SEQ ID NO:15, 17 or 20, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 35%identical to SEQ ID NO:15, at least 60% identical to SEQ ID NO:17 and/orat least 60% identical to SEQ ID NO:20.

In addition, the present inventors have identified a gene encoding a Δ6desaturase which has greater conversion efficiency for ω3 fatty acidsubstrates than for the corresponding ω6 fatty acid substrate in plantsand/or in yeast. This Δ6 desaturase also exhibits Δ8 desaturaseactivity. The use of this Δ6 desaturase or other desaturases with highspecificity for ω3 desaturated fatty acid substrates in recombinantLC-PUFA pathways in plants increases levels of EPA, DPA and DHA relativeto the use of desaturases without preference for ω3 desaturated fattyacid substrates.

Accordingly, the present invention further provides a recombinant cell,preferably a plant cell and more preferably a plant seed cell,comprising an exogenous polynucleotide encoding a fatty acid desaturasewith Δ6 desaturase activity, wherein the desaturase is furthercharacterised by having at least two, preferably all three, of thefollowing;

i) greater Δ6 desaturase activity on ALA than LA as fatty acidsubstrate, preferably in a plant cell,

ii) greater Δ6 desaturase activity on ALA-CoA as fatty acid substratethan on ALA joined to the sn-2 position of PC as fatty acid substrate,preferably in a plant cell, and

iii) Δ8 desaturase activity on ETrA, preferably in a plant cell.

The present invention further provides a recombinant cell, preferably aplant cell and more preferably a plant seed cell, comprising anexogenous polynucleotide encoding a fatty acid desaturase with Δ6desaturase activity, wherein the desaturase has greater activity on anω3 substrate than the corresponding ω6 substrate, and wherein thedesaturase has activity on ALA to produce SDA with an efficiency of atleast 5%, at least 7.5%, or at least 10% when the desaturase isexpressed from the exogenous polynucleotide in the cell, or at least 35%when expressed in a yeast cell.

In one embodiment, the desaturase has greater Δ6 desaturase activity onALA than LA as fatty acid substrate, preferably in a plant cell.

The Δ6 desaturase preferably has at least about a 2-fold greater Δ6desaturase activity, at least 3-fold greater activity, at least 4-foldgreater activity, or at least 5-fold greater activity, on ALA as asubstrate compared to LA, preferably in a plant cell.

In another embodiment, the Δ6 desaturase has greater activity on ALA-CoAas fatty acid substrate than on ALA joined to the sn-2 position of PC asfatty acid substrate, preferably in a plant cell.

The Δ6 desaturase preferably has at least about a 5-fold greater Δ6desaturase activity or at least 10-fold greater activity, on ALA-CoA asfatty acid substrate than on ALA joined to the sn-2 position of PC asfatty acid substrate, preferably in a plant cell.

The Δ6 desaturase preferably is a front-end desaturase.

In yet another embodiment, the cell according to the invention furthercomprises exogenous polynucleotides encoding;

i) a Δ6 elongase,

ii) a Δ5 desaturase,

iii) a Δ5 elongase, and

iv) optionally a Δ4 desaturase and/or an ω3 desaturase,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell.

The Δ6 desaturase in the cell of the invention preferably has nodetectable Δ5 desaturase activity on ETA.

The Δ6 desaturase preferably comprises amino acids having a sequence asprovided in SEQ ID NO:10, a biologically active fragment thereof, or anamino acid sequence which is at least 77% identical to SEQ ID NO:10.

In another embodiment, the Δ6 desaturase comprises amino acids having asequence as provided in SEQ ID NO:8, a biologically active fragmentthereof, or an amino acid sequence which is at least 67% identical toSEQ ID NO:8 and has Δ8 desaturase activity.

The present inventors have also found that recombinant cells expressingΔ9 elongase, Δ8 desaturase and Δ5 desaturase are able to moreefficiently convert fatty acid substrates to EPA, DPA and DHA.

Accordingly, the present invention further provides a recombinant cell,preferably a plant cell and more preferably a plant seed cell,comprising exogenous polynucleotides encoding;

i) a Δ9 elongase,

ii) a Δ8 desaturase,

iii) a Δ5 desaturase,

iv) optionally a Δ5 elongase, and

v) if the Δ5 elongase is present, optionally a Δ4 desaturase,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell, and wherein at least 15%, at least 20%, or at least 25%, of thetotal fatty acids in the cell comprise at least 20 carbons and at least3 carbon-carbon double bonds in their acyl chains.

In one embodiment, the sum total of ARA, EPA, DPA and DHA in the fattyacids in the cell of the invention comprises at least 15%, at least 20%,or at least 25% of the total fatty acids in the cell.

In a further embodiment, the cell according to the invention has reducedability to convert oleic acid to eicosenoic acid (C20:1) when comparedto a wild-type plant, and/or less than 5% of the oleic acid is convertedto cicoscnoic acid in the cell.

In one particular embodiment, the total fatty acid in the cell has lessthan 1% C20:1.

In a further embodiment, the cell according to the invention has reducedendogenous Δ15 desaturase activity when compared to a wild-type cell,and/or less than 10% of the LA is converted to ALA in the cell.

In one particular embodiment, the endogenous Δ15 desaturase has greateractivity on an acyl-PC substrate than on the corresponding acyl-CoAsubstrate, preferably where the acyl group is LA.

In a further embodiment, the cell of the invention further comprises anincreased conversion of GLA to SDA and/or ARA to EPA relative to thecorresponding cell lacking the exogenous polynucleotides.

In one embodiment, the amount of DHA in the fatty acids in the cell ofthe invention is at least 3%, at least 5%, or at least 10%, of the totalfatty acids in the cell.

In another embodiment, the efficiency of conversion of LA to ARA and/orALA to EPA in the cell of the invention is at least 80% or at least 90%.

In one embodiment, the Δ9 elongase comprises amino acids having asequence as provided in SEQ ID NO:22, a biologically active fragmentthereof, or an amino acid sequence which is at least 80% identical toSEQ ID NO:22.

In a further embodiment, the Δ8 desaturase comprises amino acids havinga sequence as provided in SEQ ID NO:24, a biologically active fragmentthereof, or an amino acid sequence which is at least 80% identical toSEQ ID NO:24.

In yet another embodiment, the Δ5 desaturase comprises amino acidshaving a sequence as provided in SEQ ID NO:26 or SEQ ID NO:13, abiologically active fragment thereof, or an amino acid sequence which isat least 80% identical to SEQ ID NO:26 and/or SEQ ID NO:13.

The present inventors have obtained results which indicate that a set ofgenes expressing the Δ6-desaturase, Δ6 elongase, Δ5 desaturase, Δ5elongase, and Δ4 desaturase, or a similar set of genes in particularwhere the desaturases are active on acyl-CoA substrates, can be used tosynthesise substantial levels of EPA, DPA and DHA.

Accordingly, the present invention further provides a recombinant cell,preferably a plant cell and more preferably a plant seed cell,comprising exogenous polynucleotides encoding;

i) a Δ6 elongase and/or a Δ9 elongase,

ii) a Δ6 desaturase and/or a Δ8 desaturase,

iii) a Δ5 desaturase,

iv) a Δ5 elongase,

v) a Δ4 desaturase, and

vi) optionally a diacylglycerol acyltransferase

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell, characterised by one or more or all of the following properties:

a) the efficiency of conversion of ALA to EPA, DPA or DHA is at least17.3%, or at least 23%,

b) the efficiency of conversion of ALA to DPA or DHA is at least 15.4%,or at least 21%,

c) the efficiency of conversion of ALA to DHA is at least 9.5%, or atleast 10.8%, and

d) the efficiency of conversion of EPA to DHA is at least 45%, or atleast 50%, and preferably further characterised in that at least 4% ofthe total fatty acid in the cell is DHA.

Preferably, at least 6%, at least 11% or at least 15% of the total fattyacid incorporated in triacylglycerol in the cell is DHA.

In an embodiment, DHA constitutes 20-65%, preferably, 40-65%, of thetotal of SDA, ETA, EPA, DPA and DHA in the cell.

Preferably, of the ω3 fatty acids in the cell 0.1-25% is SDA, 0.1-10% isETA, 0.1-60% is EPA, 0.1-50% is DPA and 30-95% is DHA, more preferablyof the ω3 fatty acids in the cell 0.1-25% is SDA, 0.1-10% is ETA,0.1-50% is EPA, 0.1-50% is DPA and 40-95% is DHA.

The Δ4 desaturase preferably comprises amino acids having a sequence asprovided in SEQ ID NO:73, a biologically active fragment thereof, or anamino acid sequence which is at least 80% identical to SEQ ID NO:73.

In another aspect, the present invention provides a recombinant cell,preferably a plant cell and more preferably a plant seed cell,comprising exogenous polynucleotides encoding;

i) a Δ6 elongase and/or a Δ9 elongase,

ii) a Δ6 desaturase and/or a Δ8 desaturase,

iii) a Δ5 desaturase,

iv) a Δ5 elongase, and

v) optionally a diacylglycerol acyltransferase

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell, characterised by one or more or all of the following properties:

a) the efficiency of conversion of ALA to EPA or DPA is at least 17.3%,or at least 23%, and

b) the efficiency of conversion of ALA to DPA is at least 15.4%, or atleast 21%,

and preferably further characterised in that at least 4% of the totalfatty acid in the cell is DPA.

Preferably, at least 6%, at least 11% or at least 15% of the total fattyacids incorporated in triacylglycerol in the cell is DPA.

The DPA preferably constitutes 20-65%, more preferably 40-65%, of thetotal of SDA, ETA, EPA and DPA in the cell.

Preferably, of the ω3 fatty acids in the cell 0.1-35% is SDA, 0.1-15% isETA, 0.1-60% is EPA and 30-75% is DPA, more preferably of the ω3 fattyacids in the cell 0.1-35% is SDA, 0.1-15% is ETA, 0.1-50% is EPA and40-75% is DPA.

In one embodiment, the Δ6 elongase comprises amino acids having asequence as provided in SEQ ID NO:4, a biologically active fragmentthereof, or an amino acid sequence which is at least 55% identical toSEQ ID NO:4.

In another embodiment, the Δ6 desaturase comprises amino acids having asequence as provided in SEQ ID NO:8, a biologically active fragmentthereof, or an amino acid sequence which is at least 67% identical toSEQ ID NO:8.

In one embodiment, the Δ5 desaturase comprises amino acids having asequence as provided in SEQ ID NO:26, a biologically active fragmentthereof, or an amino acid sequence which is at least 80% identical toSEQ ID NO:26.

In yet another embodiment, the Δ5 elongase comprises amino acids havinga sequence as provided in SEQ ID NO:6, a biologically active fragmentthereof, or an amino acid sequence which is at least 47% identical toSEQ ID NO:6.

In one embodiment, the diacylglycerol acyltransferase comprises aminoacids having a sequence as provided in SEQ ID NO:75 or SEQ ID NO:108, abiologically active fragment thereof, or an amino acid sequence which isat least 80% identical to SEQ ID NO:75 and/or SEQ ID NO:108.

Combinations of any two, three, four or all of the above enzymes areclearly encompassed by the invention.

In yet another embodiment, the cell, preferably a plant cell and morepreferably a plant seed cell, of the invention further comprisesexogenous polynucleotides encoding;

i) a Δ17 desaturase,

ii) a Δ15 desaturase, and/or

iii) a Δ12 desaturase

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell,

In a further embodiment, one or more or all of the desaturases expressedfrom exogenous polynucleotides in the cell of the invention have greateractivity on an acyl-CoA substrate than the corresponding acyl-PCsubstrate. In a particular embodiment, the Δ6 desaturase and the Δ5desaturase, the Δ5 desaturase and the Δ4 desaturase, the Δ6 desaturaseand the Δ4 desaturase, or all three of the Δ6 desaturase, Δ5 desaturaseand Δ4 desaturases, or additionally to each of these combinations any ofΔ17 desaturase, Δ15 desaturase and/or Δ12 desaturases have greateractivity on their acyl-CoA substrates than the corresponding acyl-PCsubstrates. In this embodiment, the other desaturases expressed fromexogenous polynucleotides in the cell may or may not have greateractivity on an acyl-CoA substrate than the corresponding acyl-PCsubstrate. As would be appreciated, the preferred acyl-CoA substrate foreach enzyme is different.

The present invention further provides a recombinant cell, preferably aplant cell and more preferably a plant seed cell, comprising anexogenous polynucleotide encoding a fatty acid elongase with Δ6 elongaseand Δ9 elongase activity, wherein the elongase has greater Δ6 elongaseactivity than Δ9 elongase activity.

In one embodiment, the elongase has an efficiency of conversion on SDAto produce ETA which is at least 50% or at least 60%, and/or anefficiency of conversion on ALA to produce ETrA which is at least 6% orat least 9%.

Preferably, the elongase has at least about 6.5 fold greater Δ6 elongaseactivity than Δ9 elongase activity.

In yet another embodiment, the elongase has no detectable Δ5 elongaseactivity.

The elongase preferably comprises amino acids having a sequence asprovided in SEQ ID NO:4, a biologically active fragment thereof, or anamino acid sequence which is at least 55% identical to SEQ ID NO:4.

In yet another embodiment, the cell further comprises exogenouspolynucleotides encoding;

i) a Δ8 desaturase and/or a Δ6 desaturase,

ii) a Δ5 desaturase,

iii) a Δ5 elongase, and

iv) optionally a Δ4 desaturase and/or an ω3 desaturase,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell.

The present invention further provides a recombinant cell, preferably aplant cell and more preferably a plant seed cell, comprising anexogenous polynucleotide encoding a fatty acid desaturase with Δ5desaturase activity, wherein the desaturase comprises amino acids havinga sequence as provided in SEQ ID NO:13, a biologically active fragmentthereof, or an amino acid sequence which is at least 53% identical toSEQ ID NO:13.

The present invention further provides a recombinant cell, preferably aplant cell and more preferably a plant seed cell, comprising anexogenous polynucleotide encoding a fatty acid elongase with Δ9 elongaseactivity, wherein the elongase comprises amino acids having a sequenceas provided in any one of SEQ ID NOs:28, 94 and 96, a biologicallyactive fragment thereof, an amino acid sequence which is at least 81%identical to SEQ ID NO:28, or an amino acid sequence which is at least50% identical to SEQ ID NO:94 and/or SEQ ID NO:96.

In an embodiment, the Δ9 elongase comprises amino acids having asequence as provided in SEQ ID NO:94 or SEQ ID NO:96, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 50%identical to SEQ ID NO:94 and/or SEQ ID NO:96, and wherein the elongasehas greater activity on an ω6 substrate than the corresponding ω3substrate More preferably, the Δ9 elongase has at least a 2 fold, morepreferably at least a 4 fold greater activity on an ω6 substrate (forexample LA) than the corresponding ω3 substrate (for example ALA).

In another aspect, the present invention provides a recombinant cellcomprising an exogenous polynucleotide encoding a diacylglycerolacyltransferase, wherein the diacylglycerol acyltransferase comprisesamino acids having a sequence as provided in SEQ ID NO:108, abiologically active fragment thereof, or an amino acid sequence which isat least 54% identical to SEQ ID NO:108.

In one embodiment of the cell according to the invention, the desaturaseand/or elongase, or multiple desaturases and/or elongases, can purifiedfrom microalga. Preferred microalgae are Pavlova spp, Pyramimonas sppand Micromonas spp.

In a preferred embodiment, the cell according to the invention is aeukaryotic cell. For example the cell may be a plant cell, a mammaliancell, an insect cell, a fungal cell or a yeast cell. The cell may be acell in tissue culture, in vitro and/or isolated.

In one embodiment, the cell is in a plant and/or is a mature plant seedcell. The plant may be in the field or harvested as a plant part, or theseed may be harvested seed.

In one particular embodiment, the plant or plant seed is an oilseedplant or an oil seed respectively.

As the skilled addressee will appreciate, one of more of the definedelongases and/or desaturases can be co-expressed in the same cell.

In a further embodiment, the cell of the invention is capable ofsynthesising long chain polyunsaturated fatty acids (LC-PUFA), whereinthe cell is derived from a cell that is not capable of synthesising saidLC-PUFA.

The present inventors have also found that co-expression of a silencingsuppressor can enhance the levels of fatty acid biosynthesis enzymes inplant cells, particularly over repeated generations from the initiallytransformed plant. Thus, in a preferred embodiment, a cell of theinvention, preferably a plant cell and more preferably a plant storageorgan cell or seed cell, comprises an exogenous polynucleotide encodinga silencing suppressor.

Preferably, the exogenous polynucleotide encoding the silencingsuppressor is operably linked to a plant storage organ specificpromoter. In an embodiment, the plant storage organ specific promoter isa seed specific promoter, or a cotyledon-specific promoter or anendosperm-specific promoter that is preferentially expressed in thedeveloping seed.

The present invention further provides a method of obtaining a cell,preferably a plant cell and more preferably a plant seed cell, capableof synthesising one or more long chain polyunsaturated fatty acids(LC-PUFAs), the method comprising

a) introducing into a cell, preferably a cell which is not capable ofsynthesising said LC-PUFA, an exogenous polynucleotide encoding a fattyacid ω3 desaturase activity, wherein the polynucleotide is operablylinked to a promoter that is capable of directing expression of saidpolynucleotide in the cell,

b) expressing the exogenous polynucleotides in the cell,

c) analysing the fatty acid composition of the cell, and

d) selecting a cell capable of desaturating at least one of ARA to EPA,DGLA to ETA, GLA to SDA, both ARA to EPA and DGLA to ETA, both ARA toEPA and GLA to SDA, or all three of these.

In one embodiment, the selected cell is a cell according to theinvention. In particular, the cell may further comprise a combination ofdesaturases and elongases as described herein.

The present invention further provides a method of obtaining a cell,preferably a plant cell and more preferably a plant seed cell, capableof synthesising one or more long chain polyunsaturated fatty acids(LC-PUFAs), the method comprising

a) introducing into a cell, preferably a cell which is not capable ofsynthesising said LC-PUFA, an exogenous polynucleotide encoding a fattyacid Δ5 elongase wherein the polynucleotide is operably linked to apromoter that is capable of directing expression of said polynucleotidein the cell,

b) expressing the exogenous polynucleotide in the cell,

c) analysing the fatty acid composition of the cell, and

d) selecting a cell wherein the Δ5 elongase has activity on EPA toproduce DPA with an efficiency of at least 60%, at least 65%, at least70% or at least 75%.

In one embodiment of the method of the invention, the selected cell is acell according to the invention. In particular, the cell may furthercomprise a combination of desaturases and elongases as described herein.

The present invention further provides a method of obtaining a cell,preferably a plant cell and more preferably a plant seed cell, capableof synthesising one or more long chain polyunsaturated fatty acids(LC-PUFAs), the method comprising

a) introducing into a cell, preferably a cell which is not capable ofsynthesising said LC-PUFA, an exogenous polynucleotide encoding a fattyacid Δ6 desaturase, wherein the polynucleotide is operably linked to apromoter that is capable of directing expression of said polynucleotidein the cell,

b) expressing the exogenous polynucleotide in the cell,

c) analysing the fatty acid composition of the cell, and

d) selecting a cell having at least two, preferably all three, of thefollowing

-   -   i) greater Δ6 desaturase activity on ALA than LA as fatty acid        substrate, preferably in a plant cell,    -   ii) greater Δ6 desaturase activity on ALA-CoA as fatty acid        substrate than on ALA joined to the sn-2 position of PC as fatty        acid substrate, preferably in a plant cell, and    -   iii) Δ6 desaturase activity on ALA and Δ8 desaturase on ETrA,        preferably in a plant cell.

The present invention further provides a method of obtaining a cell,preferably a plant cell and more preferably a plant seed cell, capableof synthesising one or more long chain polyunsaturated fatty acids(LC-PUFAs), the method comprising

a) introducing into a cell, preferably a cell which is not capable ofsynthesising said LC-PUFA, an exogenous polynucleotide encoding a fattyacid Δ6 desaturase, wherein the polynucleotide is operably linked to apromoter that is capable of directing expression of said polynucleotidein the cell,

b) expressing the exogenous polynucleotide in the cell,

c) analysing the fatty acid composition of the cell, and

d) selecting a cell with Δ6 desaturase activity which has greateractivity on an ω3 substrate than the corresponding ω6 substrate, andwith activity on ALA to produce SDA with an efficiency of at least 5%,at least 7.5%, or at least 10%, or at least 35% when expressed in ayeast cell.

In one embodiment, the selected cell is a cell according to theinvention. In particular, the cell may further comprise a combination ofdesaturases and elongases as described herein.

The present invention further provides a method of obtaining a cell,preferably a plant cell and more preferably a plant seed cell, capableof synthesising one or more long chain polyunsaturated fatty acids(LC-PUFAs), the method comprising

a) introducing into the cell, preferably a cell which is not capable ofsynthesising said LC-PUFA, exogenous polynucleotides encoding;

i) a Δ9 elongase,

ii) a Δ8 desaturase,

iii) a Δ5 desaturase,

iv) optionally a Δ5 elongase, and

v) if the Δ5 elongase is present, optionally a Δ4 desaturase,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell,

b) expressing the exogenous polynucleotides in the cell;

c) analysing the fatty acid composition of the cell, and

d) selecting a cell where at least 15%, at least 20% or at least 25% ofthe total fatty acids comprise at least 20 carbons and at least 3carbon-carbon double bonds in their acyl chains.

In one embodiment, the selected cell is a cell according to theinvention.

The present invention further provides a method of obtaining a cell,preferably a plant cell and more preferably a plant seed cell, capableof synthesising one or more long chain polyunsaturated fatty acids(LC-PUFAs), the method comprising

a) introducing into the cell, preferably a cell which is not capable ofsynthesising said LC-PUFA, exogenous polynucleotides encoding;

i) a Δ6 elongase and/or a Δ9 elongase,

ii) a Δ6 desaturase and/or a Δ8 desaturase,

iii) a Δ5 desaturase,

iv) a Δ5 elongase,

v) a Δ4 desaturase, and

vi) optionally a diacylglycerol acyltransferase,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell,

b) expressing the exogenous polynucleotides in the cell;

c) analysing the fatty acid composition of the cell, and

d) selecting a cell characterised by one or more or all of the followingproperties:

-   -   1) the efficiency of conversion of ALA to EPA, DPA or DHA is at        least 17.3%, or at least 23%;    -   2) the efficiency of conversion of ALA to DPA or DHA is at least        15.4%, or at least 21%;    -   3) the efficiency of conversion of ALA to DHA is at least 9.5%,        or at least 10.8%; and    -   4) the efficiency of conversion of EPA to DHA is at least 45%,        or at least 50%;        and preferably further characterised in that at least 4% of the        total fatty acid in the cell is DHA.

In a further aspect, the present invention provides a method ofobtaining a cell, preferably a plant cell and more preferably a plantseed cell, capable of synthesising one or more long chainpolyunsaturated fatty acids (LC-PUFAs), the method comprising

a) introducing into the cell, preferably a cell which is not capable ofsynthesising said LC-PUFA, exogenous polynucleotides encoding;

i) a Δ6 elongase and/or a Δ9 elongase,

ii) a Δ6 desaturase and/or a Δ8 desaturase,

iii) a Δ5 desaturase,

iv) a Δ5 elongase, and

v) optionally a diacylglycerol acyltransferase

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell,

b) expressing the exogenous polynucleotides in the cell;

c) analysing the fatty acid composition of the cell, and

d) selecting a cell characterised by one or more or all of the followingproperties:

a) the efficiency of conversion of ALA to EPA or DPA is at least 17.3%,or at least 23%, and

b) the efficiency of conversion of ALA to DPA is at least 15.4%, or atleast 21%,

and preferably further characterised in that at least 4% of the totalfatty acid in the cell is DPA.

In one embodiment of the method according to the invention, theexogenous polynucleotides become stably integrated into the genome ofthe cell.

In another embodiment, the method further comprises the step ofregenerating a transformed plant from the cell of step a).

In a further embodiment, the exogenous polynucleotide(s) are expressedtransiently in the cell.

In one embodiment, the cell is a leaf cell in a plant.

The present invention further provides a process for selecting a nucleicacid molecule involved in fatty acid desaturation comprising:

i) obtaining a nucleic acid molecule operably linked to a promoter, thenucleic acid molecule encoding a polypeptide which may be a fatty aciddesaturase;

ii) introducing the nucleic acid molecule into a cell in which thepromoter is active;

iii) expressing the nucleic acid molecule in the cell;

iv) analysing the fatty acid composition of the cell; and

v) selecting the nucleic acid molecule involved in fatty aciddesaturation on the basis that the polypeptide has ω3 desaturaseactivity and is capable of desaturating at least one of ARA to EPA, DGLAto ETA, GLA to SDA, both ARA to EPA and DGLA to ETA, both ARA to EPA andGLA to SDA, or all three of these.

In one embodiment of the process, the amino acid sequence of thepolypeptide is at least 35% identical to SEQ ID NO:15, at least 60%identical to SEQ ID NO:17 and/or at least 60% identical to SEQ ID NO:20.

The present invention further provides a process for selecting a nucleicacid molecule involved in fatty acid elongation comprising:

i) obtaining a nucleic acid molecule operably linked to a promoter, thenucleic acid molecule encoding a polypeptide which may be a fatty acidelongase,

ii) introducing the nucleic acid molecule into a cell in which thepromoter is active,

iii) expressing the nucleic acid molecule in the cell;

iv) analysing the fatty acid composition of the cell; and

v) selecting the nucleic acid molecule involved in fatty acid elongationon the basis that the polypeptide has Δ5 elongase activity and anefficiency of conversion on EPA to produce DPA which is at least 60%, atleast 65%, at least 70% or at least 75%.

In one embodiment, the amino acid sequence of the polypeptide is atleast 47% identical to SEQ ID NO:6.

The present invention further provides a process for selecting a nucleicacid molecule involved in fatty acid desaturation comprising:

i) obtaining a nucleic acid molecule operably linked to a promoter, thenucleic acid molecule encoding a polypeptide which may be a fatty aciddesaturase,

ii) introducing the nucleic acid molecule into a cell in which thepromoter is active,

iii) expressing the nucleic acid molecule in the cell;

iv) analysing the fatty acid composition of the cell; and

v) selecting a nucleic acid molecule involved in fatty acid desaturationon the basis that the polypeptide has Δ6 desaturase activity and atleast two, preferably all three, of the following:

a) greater Δ6 desaturase activity on ALA than LA as fatty acidsubstrate, preferably in a plant cell,

b) greater Δ6 desaturase activity on ALA-CoA as fatty acid substratethan on ALA joined to the sn-2 position of PC as fatty acid substrate,preferably in a plant cell, and

c) Δ8 desaturase activity on ALA, preferably in a plant cell.

In one embodiment of the process, the amino acid sequence of thepolypeptide is at least 77% identical to SEQ ID NO:10 and/or is at least67% identical to SEQ ID NO:8.

The present invention further provides a process for selecting a nucleicacid molecule involved in fatty acid desaturation comprising:

i) obtaining a nucleic acid molecule operably linked to a promoter, thenucleic acid molecule encoding a polypeptide which may be a fatty aciddesaturase,

ii) introducing the nucleic acid molecule into a cell in which thepromoter is active,

iii) expressing the nucleic acid molecule in the cell;

iv) analysing the fatty acid composition of the cell; and

v) selecting a nucleic acid molecule involved fatty acid desaturation onthe basis that the polypeptide has both Δ6 desaturase and Δ8 desaturaseactivities.

In one embodiment, the amino acid sequence of the polypeptide is atleast 67% identical to SEQ ID NO:8.

In a further embodiment, step (v) of the process of the inventioncomprises selecting a nucleic acid molecule encoding a desaturase activeon acyl-CoA substrates or a front-end desaturase.

The present invention further provides a combination of exogenouspolynucleotides as defined herein when used to produce a recombinantcell, express a combination of at least two fatty acid desaturases andtwo fatty acid elongases in a recombinant cell, and/or to produceLC-PUFA in a recombinant cell.

The present invention further provides a substantially purified and/orrecombinant fatty acid Δ5 elongase, wherein the elongase has activity onEPA to produce DPA with an efficiency of at least 60%, at least 65%, atleast 70% or at least 75% when expressed from an exogenouspolynucleotide in a cell.

In one embodiment, the Δ5 elongase of is characterised by any one ormore of the properties as defined herein.

The present invention further provides a substantially purified and/orrecombinant fatty acid ω3 desaturase which is capable of desaturating atleast one of ARA to EPA, DGLA to ETA, GLA to SDA, both ARA to EPA andDGLA to ETA, both ARA to EPA and GLA to SDA, or all three of these whenexpressed from an exogenous polynucleotide in a cell.

In one embodiment, the ω3 desaturase of the invention is characterisedby any one or more of the properties as defined herein.

The present invention further provides a substantially purified and/orrecombinant fatty acid Δ6 desaturase, wherein the desaturase is furthercharacterised by having at least two, preferably all three, of thefollowing;

i) greater Δ6 desaturase activity on ALA than LA as fatty acidsubstrate, preferably in a plant cell,

ii) greater Δ6 desaturase activity on ALA-CoA as fatty acid substratethan on ALA joined to the sn-2 position of PC as fatty acid substrate,preferably in a plant cell, and

iii) Δ8 desaturase activity on ETrA, preferably in a plant cell.

The present invention further provides a substantially purified and/orrecombinant fatty acid Δ6 desaturase, wherein the desaturase has greateractivity on an ω3 substrate than the corresponding ω6 substrate, andwherein the desaturase has activity on ALA to produce SDA with anefficiency of at least 5%, at least 7.5%, or at least 10% when thedesaturase is expressed from an exogenous polynucleotide in a cell, orat least 35% when expressed in a yeast cell.

In one embodiment, the Δ6 desaturase of the invention is characterisedby any one or more of the properties as defined herein.

The present invention further provides a substantially purified and/orrecombinant fatty acid Δ6 elongase and Δ9 elongase, wherein the elongasehas greater Δ6 elongase activity than Δ9 elongase activity.

In one embodiment, the Δ6 elongase and Δ9 elongase of the invention ischaracterised by any one or more of the properties as defined herein.

The present invention further provides a substantially purified and/orrecombinant fatty acid Δ5 desaturase which comprises amino acids havinga sequence as provided in SEQ ID NO:13, a biologically active fragmentthereof, or an amino acid sequence which is at least 53% identical toSEQ ID NO:13.

The present invention further provides a substantially purified and/orrecombinant fatty acid Δ9 elongase which comprises amino acids having asequence as provided in any one of SEQ ID NOs: 28, 94 and 96, abiologically active fragment thereof, an amino acid sequence which is atleast 81% identical to SEQ ID NO:28, or an amino acid sequence which isat least 50% identical to SEQ ID NO:94 and/or SEQ ID NO:96.

In an embodiment, the Δ9 elongase comprises amino acids having asequence as provided in SEQ ID NO:94 or SEQ ID NO:96, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 50%identical to SEQ ID NO:94 and/or SEQ ID NO:96, and wherein the elongasehas greater activity on an ω6 substrate than the corresponding ω3substrate.

In another aspect, the present invention provides a substantiallypurified and/or recombinant diacylglycerol acyltransferase whichcomprises amino acids having a sequence as provided in SEQ ID NO:108, abiologically active fragment thereof, or an amino acid sequence which isat least 54% identical to SEQ ID NO:108.

In an embodiment, the desaturase or elongase according to the inventioncan purified from microalga. Preferred microalgae are Pavlova spp,Pyramimonas spp and Micromonas spp.

The present invention further provides an isolated and/or exogenouspolynucleotide comprising:

i) a sequence of nucleotides selected from any one of SEQ ID NOs:3, 5,7, 9, 11, 12, 14, 16, 18, 19, 27, 29, 93, 95, 107 or 125 to 129,

ii) a sequence of nucleotides encoding a desaturase or an elongaseaccording to the invention,

iii) a sequence of nucleotides which are at least 50% identical to oneor more of the sequences set forth in SEQ ID NOs: 3, 5, 7, 9, 11, 12,14, 16, 18, 19, 27, 29, 93, 95, 107 or 125 to 129 and/or

iv) a sequence which hybridises to any one of i) to iii) under stringentconditions.

In one embodiment, the isolated and/or exogenous polynucleotidecomprises a sequence of nucleotides which is at least 57% identical toSEQ ID NO:3 and/or SEQ ID NO:126, and encodes a Δ6 elongase.

In another embodiment, the isolated and/or exogenous polynucleotidecomprises a sequence of nucleotides which is at least 50% identical toSEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and/or SEQ ID NO:19, andencodes a ω3 desaturase.

In another embodiment, the isolated and/or exogenous polynucleotidecomprises a sequence of nucleotides which is at least 50% identical toSEQ ID NO:5 and/or SEQ ID NO:128, and encodes a Δ5 elongase.

In one embodiment, the isolated and/or exogenous polynucleotidecomprises a sequence of nucleotides which is at least 75% identical toSEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and/or SEQ ID NO:125, and encodesa Δ6 desaturase.

In yet another embodiment, the isolated and/or exogenous polynucleotidecomprises a sequence of nucleotides which is at least 60% identical toSEQ ID NO:12, and encodes a Δ5 desaturase.

In another embodiment, the isolated and/or exogenous polynucleotidecomprises a sequence of nucleotides which is at least 50% identical toSEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:93 and/or SEQ ID NO:96, andencodes a Δ9 elongase.

In yet another embodiment, the isolated and/or exogenous polynucleotidecomprises a sequence of nucleotides which is at least 60% identical toSEQ ID NO:107, and encodes a diacylglycerol acyltransferase.

In one particular embodiment, the isolated and/or exogenouspolynucleotide is at least 80%, or at least 90%, or at least 95%, or atleast 99% identical to one of the sequences set forth in SEQ ID NOs: 3,5, 7, 9, 11, 12, 14, 16, 18, 19, 27, 29, 93, 95, 107 or 125 to 129.

The present invention further provides a DNA construct for integration,and/or integrated, into the genome of a plant cell, the constructcomprising a cluster of at least three open reading frames encodingproteins which modulate fatty acid synthesis in the plant cell,preferably each protein being a fatty acid desaturase or a fatty acidelongase, wherein each open reading frame having the same transcriptionorientation is separated by at least 750 bp, at least 1,000 bp or atleast 1,250 bp, and at least two of the open reading frames havedifferent transcription orientations, wherein each open reading frame isoperably linked to a promoter which is active in the plant cell and eachpromoter may independently be the same or different.

Preferably, at least two of the promoters are in the DNA construct aredifferent.

One or more or each open reading frame is preferably operably linked toa heterologous 5′ leader sequence, each of which may independently bethe same or different, wherein each heterologous 5′ leader sequenceenhances translation efficiency relative to the naturally occurring 5′leader sequence for the particular open reading frame.

In the DNA construct of the invention, the heterologous 5′ leadersequence is preferably a tobacco mosaic virus (TMV) 5′ leader sequence.

In the DNA construct according to the invention, the proteins preferablyare elongases and/or desaturases, more preferably a combination asdescribed herein.

In yet another embodiment, the DNA construct according to the inventionhas only three or four open reading frames that are translated intoproteins.

The present invention further provides a vector comprising thepolynucleotide according to the invention and/or the DNA constructaccording to the invention.

Preferably, the polynucleotide is operably linked to a promoter.

The present invention further provides a method of producing thedesaturase or elongase according to the invention, the method comprisingexpressing in a cell or cell free expression system the polynucleotideof the invention, the DNA construct of the invention and/or the vectorof the invention.

The present inventors have also surprisingly found that at least threeindependent extrachromosomal transfer nucleic acids comprising differentexogenous polynucleotides can be transiently transfected into aeukaryotic cell and the activity of each exogenous polynucleotidedetected in the cell, in combination. Thus, in another aspect thepresent invention further provides a method of transiently transfectinga eukaryotic cell with at least three different exogenouspolynucleotides, the method comprising

i) obtaining at least

-   -   a) a first bacterium comprising an extrachromosomal transfer        nucleic acid comprising a first exogenous polynucleotide,    -   b) a second bacterium comprising an extrachromosomal transfer        nucleic acid comprising a second exogenous polynucleotide, and    -   c) a third bacterium comprising an extrachromosomal transfer        nucleic acid comprising a third exogenous polynucleotide, and

ii) contacting the cell with the bacteria of step i), wherein each ofthe extrachromosomal transfer nucleic acids are transferred from thebacteria to the cell to produce the transiently transfected cell,wherein each of the exogenous polynucleotides comprises a promoter whichis active in the cell, wherein each promoter may independently be thesame or different, and wherein at least one of the exogenouspolynucleotides encodes a silencing suppressor.

Step ii) may be conducted sequentially or simultaneously with one ormore of the bacteria. For example, the cell can be contacted with thefirst bacteria, then the second bacteria and so on. In another example,the cell is contacted with each bacterium at the same time, preferablyas a mixture of the bacteria. The concentrations of the differentbacteria may be varied relative to each other or may be the same orsimilar. The bacteria may be pooled isolates, for example comprising anumber of isolates from a library of strains.

In an embodiment, the method further comprises obtaining, and thencontacting the cell with, one or more additional bacteria eachcomprising an extrachromosomal transfer nucleic acid comprisingdifferent exogenous polynucleotides. For instance, in one embodiment,the method comprises obtaining, and then contacting the cell with, afourth bacterium comprising an extrachromosomal transfer nucleic acidcomprising a fourth exogenous polynucleotide. In an additionalembodiment, the method comprises obtaining, and then contacting the cellwith, a fifth bacterium comprising an extrachromosomal transfer nucleicacid comprising a fifth exogenous polynucleotide. In an additionalembodiment, the method comprises obtaining, and then contacting the cellwith, a sixth bacterium comprising an extrachromosomal transfer nucleicacid comprising a sixth exogenous polynucleotide. In an additionalembodiment, the method comprises obtaining, and then contacting the cellwith, a seventh bacterium comprising an extrachromosomal transfernucleic acid comprising a seventh exogenous polynucleotide. In yetanother additional embodiment, the method comprises obtaining, and thencontacting the cell with, an eighth bacterium comprising anextrachromosomal transfer nucleic acid comprising an eighth exogenouspolynucleotide.

Preferably, the different exogenous polynucleotides encode different RNAmolecules and/or polypeptides.

In an embodiment, each exogenous polynucleotide encodes an enzyme whichforms part of an enzymatic pathway or is a candidate for such an enzyme.

The above aspect is particularly useful for studying polynucleotidesand/or polypeptides which form large and/or complex biological pathways.Accordingly, in a preferred embodiment, each exogenous polynucleotideencodes an enzyme, or is a candidate for such an enzyme, involved infatty acid synthesis, fatty acid modification, diacylglycerol assembly,triacylglycerol assembly, or a combination of two or more thereof

In an embodiment, one or more of the bacteria is in the form of aprotoplast. Examples of bacterium useful for the invention include, butare not limited to, Agrobacterium sp., Rhizobium sp., Sinorhizobiummeliloti, Mezorhizobium loti, Shigella flexneri, Salmonella typhimurium,Salmonella choleraesuis, Listeria monocytogenes, Escherichia coli,Yersinia pseudotuberculosis and Yersinia enterocolitica.

Examples of extrachromosomal transfer nucleic acids useful for theinvention include, but are not limited to, are P-DNA, Agrobacterium sp.T-DNA, or a combination thereof

Preferably, the cell of the above aspect is a plant cell or a mammaliancell. In an embodiment, the cell is part of a tissue or organ. Inanother embodiment, the cell is a plant cell, and the tissue or organ isa leaf, stem, root, meristem, callus, or ovule.

The present inventors have also determined that when the promoters areseed-specific promoters the expression of the exogenous polynucleotidesin leaf cells can be enhanced by co-expression of a seed specifictranscription factor such as leafy cotyledon 2, fusca3 or abscisicacid-sensitive3. Examples of leafy cotyledon 2 proteins include, but arenot limited to, those described in WO 01/70777. Thus, in a preferredembodiment, the plant cell is a plant leaf cell, at least one of thepromoters is a seed-specific promoter and at least one of the exogenouspolynucleotides encodes a seed-specific transcription such as leafycotyledon 2.

In an embodiment, none of the exogenous polynucleotides are a viralgene. In an embodiment, one or more of the exogenous polynucleotides areonly present in the extrachromosomal transfer nucleic acid as a singlecopy, not as a multimer or partial multimer of a defined nucleic acidsequence. In a further embodiment, at least one of the extrachromosomaltransfer nucleic acids does not comprise an origin of replication whichis functional in the cell, preferably not a viral origin of replicationand more preferably not the FBNYV origin of replication. In a furtherembodiment, none of the exogenous polynucleotides encodes a viralreplicase or a viral movement protein such as those described in WO2007/137788 and by Marillonnet et al. (2005).

Also provided is a method of screening a transiently transfected cellfor a desired activity, the method comprising performing the method oftransiently transfecting a eukaryotic cell with at least three exogenouspolynucleotides of the invention, and testing the cell for the desiredactivity.

The present inventors also identified that the transformation of cells,particularly plant cells, with more than six different genes can beenhanced providing the genes through different extrachromosomal transfernucleic acids. Thus, in another aspect the present invention provides amethod of transforming a eukaryotic cell with at least six differentexogenous polynucleotides, the method comprising

i) obtaining at least

-   -   a) a first bacterium comprising a first extrachromosomal        transfer nucleic acid which comprises three, four, five or six        different exogenous polynucleotides, and    -   b) a second bacterium comprising a second extrachromosomal        transfer nucleic acid different to the first which comprises        three, four, five or six different exogenous polynucleotides,

ii) contacting the cell with the bacteria of step i), and

iii) optionally selecting a cell stably transformed with the exogenouspolynucleotides of the first and second extrachromosomal transfernucleic acids, wherein each of the exogenous polynucleotides of thefirst and second extrachromosomal transfer nucleic acids are transferredfrom the bacteria to the cell to produce the transformed cell, whereineach of the exogenous polynucleotides comprises a promoter which isactive in the cell or a cell derivable therefrom, and wherein eachpromoter may independently be the same or different.

Steps i)a) and i)b) may be conducted sequentially or simultaneously withthe two bacteria. For example, the cell can be contacted with the firstbacteria and then the second bacteria. The cell contacted with thesecond bacterium may be a progeny cell or derived from the cellcontacted with the first bacterium. In another example, the cell iscontacted with both of the bacteria at the same time.

In an embodiment, the i) first extrachromosomal transfer nucleic acid,has only three to six, only three to five, only three to four, only fourto six, only four to five, or only five to six different exogenouspolynucleotides, and ii) the second extrachromosomal transfer nucleicacid, has only three to six, only three to five, only three to four,only four to six, only four to five, or only five to six differentexogenous polynucleotides.

Preferably, each of the exogenous polynucleotides encode polypeptides,and wherein each of the polypeptides are different.

In a further embodiment,

i) the first extrachromosomal transfer nucleic acid comprises twoexogenous polynucleotides independently encoding polypeptides selectedfrom the group consisting of a Δ6 desaturase, a Δ12 desaturase and a Δ15desaturase, and

ii) the second extrachromosomal transfer nucleic acid comprises anexogenous polynucleotide which encodes a polypeptide which is the thirdenzyme from the group.

Preferably, the cell is a plant cell and the method further comprisesthe step of generating a transformed plant from the stably transformedcell.

Also provided is a cell produced by the method of transientlytransfecting a eukaryotic cell with at least three exogenouspolynucleotides of the invention, or the method of transforming aeukaryotic cell with at least six different exogenous polynucleotides ofthe invention.

In yet a further aspect, the present invention provides a method ofproducing a stably transformed plant with at least six differentexogenous polynucleotides, the method comprising

i) obtaining a first stably transformed plant comprising a firstexogenous genomic region comprising three, four, five or six differentexogenous polynucleotides,

ii) obtaining a second stably transformed plant of a sexually compatiblespecies with the first and comprising a second exogenous genomic regiondifferent to the first comprising three, four, five or six differentexogenous polynucleotides,

iii) crossing the first stably transformed plant with the second stablytransformed plant, and

iv) selecting a plant produced from step iii) or a progeny thereof whichcomprises the first and second genomic regions thereby producing thestably transformed plant,

wherein each of the exogenous polynucleotides comprises a promoter whichis active in the plant, and wherein each promoter may independently bethe same or different.

In a preferred embodiment, the exogenous polynucleotides of the firstand/or second exogenous genomic regions are orientated and spaced asoutlined above for the DNA construct of the invention.

Any one promoter sequence may be present multiple times, or may be usedonly once within the first and second exogenous genomic regions, or oneor more promoters may be used multiple times and one or more otherpromoters be used only once in the first and second exogenous genomicregions. Each plant promoter may be, independently, preferentiallyactive in a tissue or organ of the plant, such as in the leaf or seed,relative to other tissues or organs. This may allow for simultaneousexpression, or overlapping expression, of all of the introduced proteincoding regions, in the plant organ or tissue. In an alternativeembodiment, one or more promoters are constitutively expressed in theplant and one or more other promoters are preferentially expressed inthe plant organ or tissue.

In an embodiment, step i) comprises producing the first stablytransformed plant by

a) contacting a plant cell with a first bacterium comprising a firstextrachromosomal transfer nucleic acid which comprises three, four, fiveor six different exogenous polynucleotides,

b) generating a stably transformed plant from the plant cell of step a),and optionally

c) producing a progeny plant from the stably transformed plant of stepb); and/or step ii) comprises producing the second stably transformedplant by

d) contacting a plant cell with a second bacterium comprising a secondextrachromosomal transfer nucleic acid which comprises three, four, fiveor six different exogenous polynucleotides,

e) generating a stably transformed plant from the plant cell of step d),and optionally

f) producing a progeny plant from the stably transformed plant of stepe).

In another aspect, the present invention provides a method of producinga stably transformed plant with at least six different exogenouspolynucleotides, the method comprising

i) obtaining a first stably transformed plant or plant part comprising afirst exogenous genomic region comprising three, four, five or sixdifferent exogenous polynucleotides,

ii) contacting a cell of the first stably transformed plant or plantpart with a bacterium comprising an extrachromosomal transfer nucleicacid which comprises three, four, five or six different exogenouspolynucleotides,

iii) producing a plant from the cell, and

iv) optionally, selecting a plant produced from step iii) whichcomprises the at least six different exogenous polynucleotides.

With regard to the step of “contacting the cell with the bacteria ofstep i)” of the above aspects, as the skilled addressee would be awarethis is preformed for a suitable time and under suitable conditions forthe extrachromosomal transfer nucleic acids to be transferred from thebacteria to the cell.

In a further aspect, the present invention provides a eukaryotic cellcomprising at least

a) a first extrachromosomal transfer nucleic acid comprising a firstexogenous polynucleotide,

b) a second extrachromosomal transfer nucleic acid comprising a secondexogenous polynucleotide, and

c) a third extrachromosomal transfer nucleic acid comprising a thirdexogenous polynucleotide.

In an embodiment, the cell further one or more additional bacteria eachcomprising an extrachromosomal transfer nucleic acid comprisingdifferent exogenous polynucleotides.

Also provided is a plant, or progeny thereof, or seed comprising a firstexogenous genomic region comprising three, four, five or six differentexogenous polynucleotides, and a second exogenous genomic regioncomprising three, four, five or six different exogenous polynucleotides.The exogenous polynucleotides of the exogenous genomic region(s) arepreferably oriented and spaced as described above for the DNA construct.

The present invention further provides a transgenic non-human organismcomprising a cell according to the invention. In an embodiment, eachcell of the organism is a cell according to the invention.

Preferably, the transgenic non-human organism is a transgenic plant,more preferably a transgenic oilseed plant to produce the oil as listedbelow. In a further embodiment, the transgenic plant comprises at leastone additional exogenous polynucleotide encoding a silencing suppressoroperably linked to a plant storage organ specific promoter, wherein theplant is phenotypically normal.

The present invention further provides a seed comprising the cellaccording to the invention or obtained from the transgenic plant of theinvention.

The present invention further provides oil produced by, or obtainedfrom, the cell according to the invention, the transgenic non-humanorganism of the invention, or the seed of the invention.

In one embodiment, the oil is obtained by extraction of oil from anoilseed.

In one embodiment, the oil is canola oil (Brassica napus, Brassica rapassp.), mustard oil (Brassica juncea), other Brassica oil, sunflower oil(Helianthus annus), linseed oil (Linum usitatissimum), soybean oil(Glycine max), safflower oil (Carthamus tinctorius), corn oil (Zeamays), tobacco oil (Nicotiana tabacum), peanut oil (Arachis hypogaea),palm oil, cottonseed oil (Gossypium hirsutum), coconut oil (Cocosnucifera), avocado oil (Persea americana), olive oil (Olea europaea),cashew oil (Anacardium occidentale), macadamia oil (Macadamiaintergrifolia), almond oil (Prunus amygdalus) or Arabidopsis seed oil(Arabidopsis thaliana).

The present invention further provides a fatty acid produced by, orobtained from, the cell according to the invention, the transgenicnon-human organism of the invention, or the seed of the invention.

The present invention further provides a method of producing oilcontaining unsaturated fatty acids, the method comprising extracting oilfrom the cell according to the invention, the transgenic non-humanorganism of the invention, or the seed of the invention.

The present invention further provides a composition comprising a cellaccording to the invention, the desaturase or elongase according to theinvention, a polynucleotide according to the invention, a DNA constructaccording to the invention, a vector of the invention, an oil accordingto the invention or a fatty acid of the invention.

The present invention further provides feedstuffs, cosmetics orchemicals comprising the cell according to the invention, the transgenicnon-human organism according to the invention, the seed according to theinvention, the oil according to the invention and/or the fatty acid ofthe invention.

The present invention further provides a method of performing adesaturase reaction, the method comprising contacting a polyunsaturatedfatty acid esterified to CoA with the desaturase of the invention.

The present invention further provides a substantially purifiedantibody, or fragment thereof, that specifically binds a desaturase orelongase of the invention.

The present invention further provides a method of treating orpreventing a condition which would benefit from a PUFA, the methodcomprising administering to a subject a cell according to the invention,the desaturase or elongase according to the invention, a polynucleotideaccording to the invention, a DNA construct according to the invention,a vector of the invention, a transgenic non-human organism according tothe invention, a seed according to the invention, an oil according tothe invention or a fatty acid of the invention and/or a feedstuff of theinvention.

In one embodiment, the condition is cardiac arrhythmia's, angioplasty,inflammation, asthma, psoriasis, osteoporosis, kidney stones, AIDS,multiple sclerosis, rheumatoid arthritis, Crohn's disease,schizophrenia, cancer, foetal alcohol syndrome, attention deficienthyperactivity disorder, cystic fibrosis, phenylketonuria, unipolardepression, aggressive hostility, adrenoleukodystophy, coronary heartdisease, hypertension, diabetes, obesity, Alzheimer's disease, chronicobstructive pulmonary disease, ulcerative colitis, restenosis afterangioplasty, eczema, high blood pressure, platelet aggregation,gastrointestinal bleeding, endometriosis, premenstrual syndrome, myalgicencephalomyelitis, chronic fatigue after viral infections or an oculardisease.

The present invention further provides use of a cell according to theinvention, the desaturase or elongase according to the invention, apolynucleotide according to the invention, a DNA construct according tothe invention, a vector of the invention, a transgenic non-humanorganism according to the invention, a seed according to the invention,an oil according to the invention or a fatty acid of the inventionand/or a feedstuff of the invention for the manufacture of a medicamentfor treating or preventing a condition which would benefit from a PUFA.

The present inventors have surprisingly found that silencing suppressorscan preferentially be expressed in plant storage organs to enhance thelevels of transgene expression in plant cells without significantlyeffecting plant development.

Accordingly, the present invention provides a plant cell comprising

i) a first exogenous polynucleotide encoding a silencing suppressor,operably linked to a plant storage organ specific promoter, and

ii) a second exogenous polynucleotide encoding an RNA molecule, operablylinked to a promoter which directs gene transcription in the plantstorage organ.

Preferably, the plant storage organ specific promoter is a seed specificpromoter such as a cotyledon specific promoter or an endosperm specificpromoter.

In an embodiment, the silencing suppressor is a viral suppressor proteinsuch as, but not limited to, P1, P19, V2, P38, P15, Pe-Po and RPV-P0.

Typically, when the viral suppressor protein is constitutively expressedin a plant the plant is phenotypically abnormal, but when the silencingsuppressor is expressed specifically in the storage organ, the plant isphenotypically normal. Examples of such viral suppressor proteinsinclude, but are not limited to, P1, P19 and P15.

In a further embodiment, the viral suppressor protein reduces microRNAaccumulation and/or microRNA guided cleavage.

The RNA molecule may be functional per se such as, but not limited to,an antisense polynucleotide, catalytic polynucleotide, dsRNA and/ormicroRNA. Alternatively, the RNA molecule may encode a polypeptide witha desired function such as, but not limited to, an enzyme involved infatty acid synthesis or modification, a seed storage protein such as forexample, a cereal glutenin or gliadin, an enzyme involved incarbohydrate synthesis or modification, secondary metabolism or apharmaceutical. Examples of pharmaceutical proteins include, but are notlimited to, antibodies as well as antibody-related molecules andfragments thereof, antigenic polypeptides which can, for example,provide immune protection against cancer, an infectious agent, acytokine such as, for example, granulocyte-macrophage colony stimulatingfactor, interferon-α, human serum albumin, and erythropoietin.

In a further embodiment, the cell comprises at least one, at least two,at least three, at least four or at least five or more additionaldifferent exogenous polynucleotides, each encoding an RNA molecule andbeing operably linked to a promoter which directs gene transcription inthe storage organ. Each exogenous polynucleotide may be operably linkedto the same promoter, different promoters or a combination thereof.

In an embodiment the exogenous polynucleotides are DNA.

In a further embodiment, the cell is in a plant storage organ such as aseed.

In a preferred embodiment, the RNA molecule is present at an increasedlevel relative to an isogenic cell lacking the first exogenouspolynucleotide. Preferably the level is increased at least 10%, at least20%, and more preferably at least 30%.

In another embodiment, at least one RNA molecule encoded by at least ofthe additional exogenous polynucleotides is present at an increasedlevel relative to an isogenic cell lacking the first exogenouspolynucleotide.

Also provided is a transgenic plant comprising a cell of the aboveaspect. In an embodiment, each cell of the plant is as defined in theabove aspect. In a particularly preferred embodiment, the plant isphenotypically normal when compared to a plant lacking said cell.

In yet another aspect provided is a plant storage organ comprising acell of the above aspect and/or obtained from the transgenic plantdefined above.

In an embodiment, the plant storage organ is a seed.

In a further aspect, provided is a method of obtaining a phenotypicallynormal plant having increased levels of an RNA molecule in its storageorgan, comprising

a) introducing into a plant cell

-   -   i) a first exogenous polynucleotide encoding a silencing        suppressor operably linked to a plant storage organ specific        promoter, and    -   ii) a second exogenous polynucleotide encoding an RNA molecule        operably linked to a promoter which directs gene transcription        in the plant storage organ,

b) regenerating a transformed plant from the cell of step a),

c) growing the transformed plant until it produces storage organs,

d) determining the level of the RNA molecule in the storage organ, and

e) selecting a plant which is phenotypically normal, and wherein the RNAmolecule is present at an increased level in the storage organ relativeto a corresponding storage organ lacking the first exogenouspolynucleotide.

In yet a further aspect, provided is a method of obtaining aphenotypically normal plant having stabilized expression of an RNAmolecule in its storage organ, comprising

a) introducing into a plant cell

-   -   i) a first exogenous polynucleotide encoding a silencing        suppressor operably linked to a plant storage organ specific        promoter, and    -   ii) a second exogenous polynucleotide encoding an RNA molecule        operably linked to a promoter which directs gene transcription        in the plant storage organ,

b) regenerating a transformed plant from the cell of step a),

c) producing a third generation progeny plant which comprises thestorage organ from the plant of step b), and

d) selecting a third generation progeny plant wherein the RNA moleculeis present in the storage organ at a level which is at least 90% of thelevel in a storage organ of a previous generation of the plant.

Preferably, the exogenous polynucleotides of the above aspects arestably integrated into the genome of the cell.

In yet another aspect, the present invention provides a method ofstabilising expression of an RNA molecule in a storage organ of atransgenic plant, comprising

i) expressing a first exogenous polynucleotide encoding a silencingsuppressor operably linked to a plant storage organ specific promoter,and

ii) expressing a second exogenous polynucleotide encoding an RNAmolecule operably linked to a promoter which directs gene transcriptionin the plant storage organ,

wherein the transgenic plant is at least a third generation progenyplant obtained from a parental plant transformed with the exogenouspolynucleotides, and wherein the RNA molecule is present in the storageorgan of the plant at a level which is at least 90% of the level in astorage organ of a previous generation of the plant.

In an embodiment, the plant is grown in the field.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. Aerobic DHA biosynthesis pathways.

FIG. 2. The various acyl exchange enzymes which transfer fatty acidsbetween PC, CoA pools, and TAG pools. Adapted from Singh et al. (2005).

FIG. 3. Multiple alignment between the Micromonas CS-0170 Δ6-elongaseand related genes. AAV33630, C20-polyunsaturated fatty acid elongatingenzyme [Pavlova sp. CCMP459]; AAY15135, elongase 1 [Pavlova salina];ABR67690, C20 elongase [Pavlova viridis]; AAV67797, polyunsaturatedfatty acid elongase 1 [Ostreococcus tauri]; CAL55414, polyunsaturatedfatty acid elongase 2 (ISS) [Ostreococcus tauri]; XP_001419791,predicted protein [Ostreococcus lucimarinus CCE9901]; MicCS0170-d6E,Micromonas CS-0170 Δ6-elongase (this work); AAV67800, polyunsaturatedfatty acid elongase 2 [Thalassiosira pseudonana]; XP_001416454,predicted protein [Ostreococcus lucimarinus CCE9901]; ABC18313,polyunsaturated fatty acid elongase 1 [Thraustochytrium sp. FJN-10];AAV67799, polyunsaturated fatty acid elongase 1 [Thalassiosirapseudonana]; AAW70157, delta-6-elongase [Phaeodactylum tricornutum]; ABC18314, polyunsaturated fatty acid elongase 2 [Thraustochytrium sp.FJN-10]; CAD58540, unnamed protein product [Isochrysis galbana].

FIG. 4. Multiple alignment between the Pyramimonas CS-0140 Δ6-elongaseand related genes. AAL84174, polyunsaturated fatty acid specificelongation enzyme 1 [Physcomitrella patens]; AAT85662, polyunsaturatedfatty acid elongase [Marchantia polymorpha]; AAV67797, polyunsaturatedfatty acid elongase 1 [Ostreococcus tauri]; AB094747, predicted protein[Ostreococcus lucimarinus CCE9901]; Pyrco-d6E, Pyramimonas CS-0140Δ6-elongase (this work); ABC18313, polyunsaturated fatty acid elongase 1[Thraustochytrium sp. FJN-10]; BAF97073, polyunsaturated fatty acidelongation enzyme [Mortierella alpina]; XP_001567488, long chainpolyunsaturated fatty acid elongation enzyme-like protein [Leishmaniabraziliensis MHOM/BR/75/M2904]; BAE71129, delta5-elongase [Marchantiapolymorpha]; XP_001779105; predicted protein [Physcomitrella patens].

FIG. 5. Multiple alignment between the Pyramimonas CS-0140 Δ5-elongaseand related genes. AΔ152204, Elov14 protein [Danio rerio]; CAG01780,unnamed protein product [Tetraodon nigroviridis]; AAV67800,polyunsaturated fatty acid elongase 2 [Thalassiosira pseudonana];AAV33630, C20-polyunsaturated fatty acid elongating enzyme [Pavlova sp.CCMP459]; ABR67690, C20 elongase [Pavlova viridis]; AAY15135, elongase 1[Pavlova salina]; AAV67798, polyunsaturated fatty acid elongase 2[Ostreococcus tauri]; AB098084, predicted protein [Ostreococcuslucimarinus CCE9901]; Pyrco-d5E, Pyramimonas CS-0140 Δ5-elongase (thiswork).

FIG. 6. Multiple alignment between the Micromonas CCMP1545 Δ6-desaturaseand related genes. AAM09687, Δ5-fatty acid desaturase [Thraustochytriumsp. ATCC21685]; AAV33631, Δ4-desaturase [Isochrysis galbana]; AAW70159,Δ6-desaturase [Ostreococcus tauri]; AB099366, predicted protein[Ostreococcus lucimarinus CCE9901]; Mic-d6D, Micromonas CCMP 1545Δ6-desaturase (this work); ABF58685, Δ5-desaturase [Perkinsus marinas];ABL96295, Δ5-desaturase [Pavlova salina].

FIG. 7. Multiple alignment between the Ostreococcus lucimarinusΔ6-desaturase and related genes. AAM09687, Δ5-fatty acid desaturase[Thraustochytrium sp. ATCC21685]; AAR27297, Δ6-desaturase [Amylomycesrouxii]; AAS93682, Δ6-fatty acid desaturase [Rhizopus oryzae]; AAV33631,Δ4-desaturase [Isochrysis galbana]; AAW70159, Δ6-desaturase[Ostreococcus tauri]; Ostlu-d6D, Ostreococcus lucimarinus Δ6-desaturase(this work); ABF58685, Δ5-desaturase [Perkinsus marinus]; ABL96295,Δ5-desaturase [Pavlova salina]; EDQ92231, predicted protein [Monosigabrevicollis MX1]; CAM41728, fatty acid desaturase, putative [Leishmaniabraziliensis]; CAM65683, fatty acid desaturase, putative [Leishmaniainfantum].

FIG. 8 Multiple alignment between the Pyramimonas CS-0140 Δ5-desaturaseand related genes. AAM09687, Δ5-fatty acid desaturase [Thraustochytriumsp. ATCC21685]; Pyrco-d5D, Pyramimonas CS-0140 Δ5-desaturase (thiswork); AAT85661, Δ6-fatty acid desaturase [Marchantia polymorpha];AAX14505, Δ6-fatty acid desaturase [Thalassiosira pseudonana]; ABP49078,Δ6-fatty acid desaturase [Phaeodactylum tricornutum]; AAW70159,Δ6-desaturase [Ostreococcus tauri]; XP_001421073, predicted protein[Ostreococcus lucimarinus CCE9901]; ABF58685, Δ5-desaturase [Perkinsusmarinus]; CAJ07076, fatty acid desaturase, putative [Leishmania major];ABL96295, Δ5-desaturase [Pavlova salina]; EDQ92231, predicted protein[Monosiga brevicollis MX1].

FIG. 9. Multiple alignment of the Ostreococcus tauri (Ot) (SEQ IDNO:30), Ostreococcus lucimarinus (O1) (SEQ ID NO:10) and Micromonas (M)CCMP1545 Δ6-desaturase protein sequences (SEQ ID NO:8).

FIG. 10. Phylogenetic tree showing the relationship between variousdesaturases. Pavsa-d5D=Pavlova salina Δ5-desaturase (ABL96295);Thr21685-d5D=Thraustochytrium sp. ATCC21685 Δ5-desaturase (AAM09687);Mic1545-d6D=Micromonas CCMP1545 Δ6-desaturase (this work);Ostta-d6D=Ostreococcus tauri Δ6-desaturase (AAW70159);Ostlu-d6D=Ostreococcus lucimarinus Δ6-desaturase (this work);Galga-d6D=Gallus gallus Δ6-desaturase (XP_421053); Homsa-d6D=Homosapiens Δ6-desaturase (AAG23121); Musmu-d6D=Mus musculus Δ6-desaturase(NP 062673); Danre-d5/6D=Danio rerio Δ5-/Δ6-desaturase (AAG25710);Spaau-d6D=Sparus aurata putative Δ6-desaturase (AAL17639);Scoma-d6D=Scophthalmus maximus Δ6-desaturase (AAS49163);Oncmy-d6D=Oncorhynchus mykiss Δ6-desaturase (AAK26745); Salsa-d5D=Salmosalar Δ5-desaturase (AAL82631); Prifa-d6D=Primula farinosa Δ6-desaturase(AAP23034); Euggr-d6D=Euglena gracilis Δ6-desaturase; Borof-d6D=Boragoofficianalis Δ6-desaturase (AAC49700); Caeel-d6D=Caenorhabditis elegansΔ6-desaturase (AAC15586); Rhior-d6D=Rhizopus oryzae Δ6-desaturase(AAS93682); Moral-d6D=Mortierella alpina Δ6-desaturase (AAF08685);Moris-d6D=Mortierella isabellina Δ6-desaturase (AAL73948);Marpo-d6D=Marchantia polymorpha Δ6-desaturase (AAT85661);Cerpu-d6D=Ceratodon purpureus Δ6-desaturase (CAB94993); Phatr-d6DPhaeodactylum tricornutum Δ6-desaturase (AAL92563);Thaps-d6D=Thalassiosira pseudonana Δ6-desaturase (AAX14505);Pavsa-d8D=Pavlova salina Δ8-desaturase (ABL96296);Phatr-d5D=Phaeodactylum tricornutum Δ5-desaturase (AAL92562);Marpo-d5D=Marchantia polymorpha Δ5-desaturase (AAT85663);Moral-d5D=Mortierella alpina Δ5-desaturase (AAR28035);Dicdi-d5D=Dictyostelium discoideum Δ5-desaturase (BAΔ37090).

FIG. 11. GC results from T2 Arabidopsis seed transformed with thelinP-mic1545-d6D-linT construct. SDA and GLA levels are shown forindividual events 1-19, with the ratio of ω3 to ω6 conversionefficiencies displayed above each column. The M. pusilla Δ6-desaturaseshows clear preference for the ω3 substrate.

FIG. 12. Conversion efficiencies of enzymes constituting the EPApathways infiltrated into N. benthamiana. The EPA pathways contain aΔ6-desaturase (Echium plantagineum Δ6-desaturase, Ostreococcus tauriΔ6-desaturase or Micromonas pusilla Δ6-desaturase), the Pyramimonascordata Δ6-elongase and the Pavlova salina Δ5-desaturase. Panel a. showsthe ω3 pool conversion efficiencies for each pathway; b. contains directcomparisons between the E. plantagineum pathway, the M. pusilla pathwayand a pathway containing both these desaturases; c. contains directcomparisons between the O. tauri pathway, the M. pusilla pathway and apathway containing both acyl-CoA desaturases.

FIG. 13. GC and GC-MS confirmation of the production of EPA in Nicotianabenthamiana by a transiently-expressed Micromonas RCC299 ω3 desaturase.

FIG. 14. Map of the binary vector pJP10lacq showing the key features ofthe binary vector including the promoter orientations, TMV leadersequence locations, spacer region locations and unique cloning sites forgene insertions. NosT=NOS terminator, FP 1=truncated napin terminator,LininT=Linin terminator.

FIG. 15. Map of the binary vector pJP107.

FIG. 16. Determination of the Agrobacterium concentration required toachieve near-maximal gene activity in the leaf-based assay. Isochrysisgalbana Δ9-elongase (IgΔ9elo) activity in N. benthamiana afterinfiltration with varying culture densities of Agrobacterium AGL1containing the binary expression construct IgΔ9elo. Co-infiltrated P19was set at a concentration of OD_(600nm) 0.4. The y-axis displays thesum of both IgΔ9elo activities to produce EDA and ETrA.

FIG. 17. Comparison of transgenic expression of LC-PUFA pathways usingeither transient or stable expression in leaves. Conversion efficienciesare based on total fatty acid profiles. ^(a)results extracted from (Qiet al., 2004); ^(b)results extracted from (Robert et al., 2005).

FIG. 18. Metabolic tailoring in Nicotiana benthamiana. Panel a. is a gaschromatography (GC) trace of fatty acid methyl esters (FAME) producedfrom tuna oil which contains only a small amount of EPA but a largeamount of DHA. Panels b. and c. are GC traces of FAME derived from theTAG fraction of N. benthamiana leaf tissue transiently transformed withsingle-gene CaMV 35S binary constructs containing the Micromonas pusillaΔ6-desaturase, Pyramimonas cordata Δ6-elongase, Pavlova salinaΔ5-desaturase, P. cordata Δ5-elongase (b.) or P. salina Δ5-elongase (c.)and the P. salina Δ4-desaturase. The accumulation of EPA in the sampleusing the P. salina Δ5-elongase demonstrates the manner in whichmetabolic pathways can be tailored by careful selection of a single genein the pathway.

FIG. 19. Map of the region of vector pJP3075 comprising transgenes.

FIG. 20. Map of the region of vector pJP3059 comprising transgenes.

FIG. 21. Map of the region of vector pJP3060 comprising transgenes.

FIG. 22. Map of the region of vector pJP3115 comprising transgenes.

FIG. 23. Map of the region of vector pJP3116 comprising transgenes.

KEY TO THE SEQUENCE LISTING

-   SEQ ID NO:1—Open reading frame encoding Micromonas CS-0170    Δ6-elongase.-   SEQ ID NO:2—Micromonas CS-0170 Δ6-elongase.-   SEQ ID NO:3—Open reading frame encoding Pyramimonas CS-0140    Δ6-elongase/Δ9-elongase.-   SEQ ID NO:4—Pyramimonas CS-0140 Δ6-elongase/Δ9-elongase.-   SEQ ID NO:5—Open reading frame encoding Pyramimonas CS-0140    Δ5-elongase.-   SEQ ID NO:6—Pyramimonas CS-0140 Δ5-elongase.-   SEQ ID NO:7—Open reading frame encoding Micromonas CCMP1545    Δ6-desaturase/Δ8-desaturase.-   SEQ ID NO:8—Micromonas CCMP1545 Δ6-desaturase/Δ8-desaturase.-   SEQ ID NO:9—Open reading frame encoding Ostreococcus lucimarinus    Δ6-desaturase.-   SEQ ID NO:10—Ostreococcus lucimarinus Δ6-desaturase.-   SEQ ID NO:11—Codon-optimized open reading frame for expression of    Ostreococcus lucimarinus Δ6-desaturase in plants.-   SEQ ID NO:12—Open reading frame encoding Pyramimonas CS-0140    Δ5-desaturase.-   SEQ ID NO:13—Pyramimonas CS-0140 Δ5-desaturase.-   SEQ ID NO:14—Partial open reading frame encoding Micromonas CS-0170    ω3-desaturase.-   SEQ ID NO:15—Partial Micromonas CS-0170 ω3-desaturase.-   SEQ ID NO:16—Open reading frame encoding Micromonas RCC299    ω3-desaturase-   SEQ ID NO:17—Micromonas RCC299 ω3-desaturase.-   SEQ ID NO:18—Codon-optimized open reading frame for expression of    Micromonas RCC299 ω3-desaturase in plants.-   SEQ ID NO:19—Open reading frame encoding Micromonas CCMP1545    ω3-desaturase-   SEQ ID NO:20—Micromonas CCMP1545 ω3-desaturase.-   SEQ ID NO:21—Open reading frame encoding Isochrysis galbana    Δ9-elongase.-   SEQ ID NO:22—Isochrysis galbana Δ9-elongase.-   SEQ ID NO:23—Open reading frame encoding Pavlova salina    Δ8-desaturase.-   SEQ ID NO:24—Pavlova salina Δ8-desaturase.-   SEQ ID NO:25—Open reading frame encoding Pavlova salina    Δ5-desaturase.-   SEQ ID NO:26—Pavlova salina Δ5-desaturase.-   SEQ ID NO:27—Open reading frame encoding Emiliania huxleyi CCMP1516    Δ9 elongase.-   SEQ ID NO:28—Emiliania huxleyi CCMP1516 Δ9 elongase.-   SEQ ID NO:29—Codon-optimized open reading frame for expression of    Emiliania huxleyi Δ9 elongase in plants.-   SEQ ID NO:30—Ostreococcus tauri Δ6-desaturase.-   SEQ ID NO:31—Elongase consensus domain 1.-   SEQ ID NO:32—Elongase consensus domain 2.-   SEQ ID NO:33—Elongase consensus domain 3.-   SEQ ID NO:34—Elongase consensus domain 4.-   SEQ ID NO:35—Elongase consensus domain 5.-   SEQ ID NO:36—Elongase consensus domain 6.-   SEQ ID NO:37—Desaturase consensus domain 1.-   SEQ ID NO:38—Desaturase consensus domain 2.-   SEQ ID NO:39—Desaturase consensus domain 3.-   SEQ ID NO:40—Desaturase consensus domain 4.-   SEQ ID NOs:41-71 and 78-92—Oligonucleotide primers.-   SEQ ID NO:72—Open reading frame encoding Pavlova salina    Δ4-desaturase.-   SEQ ID NO:73—Pavlova salina Δ4-desaturase.-   SEQ ID NO:74—Open reading frame encoding Arabidopsis thaliana    diacylglycerol acyltransferase 1.-   SEQ ID NO:75—Arabidopsis thaliana diacylglycerol acyltransferase 1.-   SEQ ID NO:76—Elongase consensus domain 7.-   SEQ ID NO:77—Elongase consensus domain 8.-   SEQ ID NO:93—Open reading frame encoding Pavlova pinguis    Δ9-elongase.-   SEQ ID NO:94—Pavlova pinguis Δ9-elongase.-   SEQ ID NO:95—Open reading frame encoding Pavlova salina Δ9-elongase.-   SEQ ID NO:96—Pavlova salina Δ9-elongase.-   SEQ ID NO:97—P19 viral suppressor.-   SEQ ID NO:98—V2 viral suppressor.-   SEQ ID NO:99—P38 viral suppressor.-   SEQ ID NO:100—Pe-P0 viral suppressor.-   SEQ ID NO:101—RPV-P0 viral suppressor.-   SEQ ID NO:102—Open reading frame encoding P19 viral suppressor.-   SEQ ID NO:103—Open reading frame encoding V2 viral suppressor.-   SEQ ID NO:104—Open reading frame encoding P38 viral suppressor.-   SEQ ID NO:105—Open reading frame encoding Pe-P0 viral suppressor.-   SEQ ID NO:106—Open reading frame encoding RPV-P0 viral suppressor.-   SEQ ID NO:107—Codon optimized open reading frame encoding Micromonas    CCMP1545 diacylglycerol acyltransferase 2.-   SEQ ID NO:108—Micromonas CCMP1545 diacylglycerol acyltransferase 2.-   SEQ ID NO's 109-124—Transfer nucleic acid border sequences.-   SEQ ID NO:125—Codon-optimized open reading frame for expression of    Micromonas CCMP1545 Δ6 desaturase/Δ8 desaturase in plants.-   SEQ ID NO:126—Codon-optimized open reading frame for expression of    Pyramimonas CS-0140 Δ6 elongase/Δ9 elongase in plants (truncated at    3′ end and encoding functional elongase).-   SEQ ID NO:127—Codon-optimized open reading frame for expression of    Pavlova salina Δ5 desaturase in plants.-   SEQ ID NO:128—Codon-optimized open reading frame for expression of    Pyramimonas CS-0140 Δ5 elongase in plants.-   SEQ ID NO:129—Codon-optimized open reading frame for expression of    Pavlova salina Δ4 desaturase in plants.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, fatty acid synthesis, transgenic plants, proteinchemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), F. M. Ausubel et al.(editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors), Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors), Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

Selected Definitions

As used herein, the term “fatty acid” refers to a carboxylic acid (ororganic acid), often with a long aliphatic tail, either saturated orunsaturated. Typically fatty acids have a carbon-carbon bonded chain ofat least 8 carbon atoms in length, more preferably at least 12 carbonsin length. Most naturally occurring fatty acids have an even number ofcarbon atoms because their biosynthesis involves acetate which has twocarbon atoms. The fatty acids may be in a free state (non-esterified) orin an esterified form such as part of a triglyceride, diacylglyceride,monoacylglyceride, acyl-CoA (thio-ester) bound or other bound form. Thefatty acid may be esterified as a phospholipid such as aphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol or di phosphatidyl glycerolforms.

“Saturated fatty acids” do not contain any double bonds or otherfunctional groups along the chain. The term “saturated” refers tohydrogen, in that all carbons (apart from the carboxylic acid [—COOH]group) contain as many hydrogens as possible. In other words, the omega(ω) end contains 3 hydrogens (CH3-) and each carbon within the chaincontains 2 hydrogens (—CH2-).

“Unsaturated fatty acids” are of similar form to saturated fatty acids,except that one or more alkene functional groups exist along the chain,with each alkene substituting a singly-bonded “—CH2-CH2-” part of thechain with a doubly-bonded “—CH═CH—” portion (that is, a carbon doublebonded to another carbon). The two next carbon atoms in the chain thatare bound to either side of the double bond can occur in a cis or transconfiguration.

As used herein, the term “monounsaturated fatty acid” refers to a fattyacid which comprises at least 12 carbon atoms in its carbon chain andonly one alkene group (carbon-carbon double bond) in the chain. As usedherein, the terms “polyunsaturated fatty acid” or “PUFA” refer to afatty acid which comprises at least 12 carbon atoms in its carbon chainand at least two alkene groups (carbon-carbon double bonds).

As used herein, the terms “long-chain polyunsaturated fatty acid” and“LC-PUFA” refer to a fatty acid which comprises at least 20 carbon atomsin its carbon chain and at least two carbon-carbon double bonds, andhence include VLC-PUFAs. As used herein, the terms “very long-chainpolyunsaturated fatty acid” and “VLC-PUFA” refer to a fatty acid whichcomprises at least 22 carbon atoms in its carbon chain and at leastthree carbon-carbon double bonds. Ordinarily, the number of carbon atomsin the carbon chain of the fatty acids refers to an unbranched carbonchain. If the carbon chain is branched, the number of carbon atomsexcludes those in sidegroups. In one embodiment, the long-chainpolyunsaturated fatty acid is an ω3 fatty acid, that is, having adesaturation (carbon-carbon double bond) in the third carbon-carbon bondfrom the methyl end of the fatty acid. In another embodiment, thelong-chain polyunsaturated fatty acid is an ω6 fatty acid, that is,having a desaturation (carbon-carbon double bond) in the sixthcarbon-carbon bond from the methyl end of the fatty acid. In a furtherembodiment, the long-chain polyunsaturated fatty acid is selected fromthe group consisting of; arachidonic acid (ARA, 20:4Δ5,8,11,14; ω6),eicosatetraenoic acid (ETA, 20:4Δ8,11,14,17, ω3) eicosapentaenoic acid(EPA, 20:5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA,22:5Δ7,10,13,16,19, ω3), or docosahexaenoic acid (DHA,22:6Δ4,7,10,13,16,19, ω3). The LC-PUFA may also be dihomo-γ-linoleicacid (DGLA) or eicosatrienoic acid (ETrA, 20:3Δ11,14,17, ω3). It wouldreadily be apparent that the LC-PUFA that is produced according to theinvention may be a mixture of any or all of the above and may includeother LC-PUFA or derivatives of any of these LC-PUFA. In a preferredembodiment, the ω3 fatty acid is EPA, DPA, and/or DHA, preferably, EPAand/or DPA, or preferably DPA and/or DHA.

Furthermore, as used herein the terms “long-chain polyunsaturated fattyacid” and “very long-chain polyunsaturated fatty acid” refer to thefatty acid being in a free state (non-esterified) or in an esterifiedform such as part of a triglyceride, diacylglyceride, monoacylglyceride,acyl-CoA bound or other bound form. The fatty acid may be esterified asa phospholipid such as a phosphatidylcholine (PC),phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidylinositol or diphosphatidylglycerol forms. Thus, the LC-PUFAmay be present as a mixture of forms in the lipid of a cell or apurified oil or lipid extracted from cells, tissues or organisms. Inpreferred embodiments, the invention provides oil comprising at least75% or 85% triacylglycerols, with the remainder present as other formsof lipid such as those mentioned, with at least said triacylglycerolscomprising the LC-PUFA. The oil may be further purified or treated, forexample by hydrolysis with a strong base to release the free fatty acid,or by fractionation, distillation or the like.

The desaturase, elongase and acyl transferase proteins and genesencoding them that may be used in the invention are any of those knownin the art or homologues or derivatives thereof. Examples of such genesand encoded protein sizes are listed in Table 1. The desaturase enzymesthat have been shown to participate in LC-PUFA biosynthesis all belongto the group of so-called “front-end” desaturases.

TABLE 1 Cloned genes involved in LC-PUFA biosynthesis Protein sizeEnzyme Type of organism Species Accession Nos. (aa's) ReferencesΔ4-desaturase Protist Euglena gracilis AY278558 541 Meyer et al., 2003Algae Pavlova lutherii AY332747 445 Tonon et al., 2003 Isochrysisgalbana AAV33631 433 Pereira et al., 2004b Pavlova salina AAY15136 447Zhou et al., 2007 Thraustochytrid Thraustochytrium aureum AAN75707 515N/A AAN75708 AAN75709 AAN75710 Thraustochytrium sp. AAM09688 519 Qiu etal. 2001 ATCC21685 Δ5-desaturase Mammals Homo sapiens AF199596 444 Choet al., 1999b Leonard et al., 2000b Nematode Caenorhabditis elegansAF11440, 447 Michaelson et al., 19981); NM_069350 Watts and Browse,1999b Fungi Mortierella alpina AF067654 446 Michaelson et al., 1998a;Knutzon et al., 1998 Pythium irregulare AF419297 456 Hong et al., 2002aDictyostelium discoideum AB022097 467 Saito et al., 2000 Saprolegniadiclina 470 WO02081668 Diatom Phaeodactylum tricornutum AY082392 469Domergue et al., 2002 Algae Thraustochytrium sp AF489588 439 Qiu et al.,2001 Thraustochytrium aureum 439 WO02081668 Isochlysis galbana 442WO02081668 Moss Marchantia polymorpha AY583465 484 Kajikawa et al., 2004Δ6-desaturase Mammals Homo sapiens NM_013402 444 Cho et al., 1999a;Leonard et al., 2000 Mus musculus NM_019699 444 Cho et al., 1999aNematode Caenorhabditis elegans Z70271 443 Napier et al., 1998 PlantsBorago officinales U79010 448 Sayanova et al., 1997 Echium AY055117Garcia-Maroto et al., 2002 AY055118 Primula vialii AY234127 453 Sayanovaet al., 2003 Anemone leveillei AF536525 446 Whitney et al., 2003 MossesCeratodon purpureus AJ250735 520 Sperling et al., 2000 Marchantiapolymorpha AY583463 481 Kajikawa et al., 2004 Physcomitrella patensCAA11033 525 Girke et al., 1998 Fungi Mortierella alpina AF110510 457Huang et al., 1999; AB020032 Sakuradani et al., 1999 Pythium irregulareAF419296 459 Hong et al., 2002a Mucor circinelloides AB052086 467 NCBI*Rhizopus sp. AY320288 458 Zhang et al., 2004 Saprolegnia diclina 453WO02081668 Diatom Phaeodactylum tricornutum AY082393 477 Domergue etal., 2002 Bacteria Synechocystis L11421 359 Reddy et al., 1993 AlgaeThraustochytrium aureum 456 WO02081668 Bifunctional Fish Danio rerioAF309556 444 Hastings et al., 2001 Δ5/Δ6 desaturase C20 Δ8- AlgaeEuglena gracilis AF139720 419 Wallis and Browse, 1999 desaturase PlantsBorago officinales AAG43277 446 Sperling et al., 2001 Δ6-elongaseNematode Caenorhabditis elegans NM_069288 288 Beaudoin at al., 2000Mosses Physcomitrella patens AF428243 290 Zank et al., 2002 Marchantiapolymorpha AY583464 290 Kajikawa et al., 2004 Fungi Mortierella alpinaAF206662 318 Parker-Barnes et al., 2000 Algae Pavlova lutheri** 501 WO03078639 Thraustochytrium AX951565 271 WO 03093482 Thraustochytriun sp**AX214454 271 WO 0159128 PUFA- Mammals Homo sapiens AF231981 299 Leonardet al., 2000b; elongase Leonard et al., 2002 Rattus norvegicus AB071985299 Inagaki et al., 2002 Rattus norvegicus** AB071986 267 Inagaki etal., 2002 Mus musculus AF170907 279 Tvrdik et al., 2000 Mus musculusAF170908 292 Tvrdik et al., 2000 Fish Danio rerio AF532782 291 (282)Agaba et al., 2004 Danio rerio** NM 199532 266 Lo et al., 2003 WormCaenorhabditis elegans Z68749 309 Abbott et al 1998 Beaudoin et al 2000Algae Thraustochytrium ameum** AX464802 272 WO 0208401-A2 Pavlovalutheri** 320 WO 03078639 Δ9-elongase Algae Isochrysis galbana AF390174263 Qi et al., 2002 Euglena gracilis 258 WO 08/128241 Δ5-elongase AlgaeOstreococcus tauri AAV67798 300 Meyer et al., 2004 Pyramimonas cordata268 this work Pavlova sp. CCMP459 AAV33630 277 Pereira et al., 2004bPavlova salina AAY15135 302 Robert et al., 2009 Diatom Thalassiosirapseudonana AAV67800 358 Meyer et al., 2004 Fish Oncorhynchus mykissCAM55862 295 WO 06/008099 Moss Marchantia polymorpha BAE71129 348Kajikawa et al., 2006 *http://www.ncbi.nlm.nih.gov/ **Function notproven/not demonstrated

As used herein, the term “front-end desaturase” refers to a member of aclass of enzymes that introduce a double bond between the carboxyl groupand a pre-existing unsaturated part of the acyl chain of lipids, whichare characterized structurally by the presence of an N-terminalcytochrome b5 domain, along with a typical fatty acid desaturase domainthat includes three highly conserved histidine boxes (Napier et al.,1997).

Activity of any of the elongases or desaturases for use in the inventionmay be tested by expressing a gene encoding the enzyme in a cell suchas, for example, a yeast cell or a plant cell, and determining whetherthe cell has an increased capacity to produce LC-PUFA compared to acomparable cell in which the enzyme is not expressed.

In one embodiment the desaturase and/or elongase for use in theinvention can purified from a microalga.

Whilst certain enzymes are specifically described herein as“bifunctional”, the absence of such a term does not necessarily implythat a particular enzyme does not possess an activity other than thatspecifically defined.

Desaturases

As used herein, the term “desaturase” refers to an enzyme which iscapable of introducing a carbon-carbon double bond into the acyl groupof a fatty acid substrate which is typically in an esterified form suchas, for example, fatty acid CoA esters. The acyl group may be esterifiedto a phospholipid such as phosphatidylcholine (PC), or to acyl carrierprotein (ACP), or in a preferred embodiment to CoA. Desaturasesgenerally may be categorized into three groups accordingly. In oneembodiment, the desaturase is a front-end desaturase.

As used herein, a “Δ4 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the4^(th) position from the carboxyl end of a fatty acid substrate. The “Δ4desaturase” is at least capable of converting DPA to DHA. Thedesaturation step to produce DHA from DPA is catalysed by a Δ4desaturase in organisms other than mammals, and a gene encoding thisenzyme has been isolated from the freshwater protist species Euglenagracilis and the marine species Thraustochytrium sp. (Qiu et al., 2001;Meyer et al., 2003). In one embodiment, the Δ4 desaturase comprisesamino acids having a sequence as provided in SEQ ID NO:73, abiologically active fragment thereof, or an amino acid sequence which isat least 80% identical to SEQ ID NO:73.

As used herein, a “Δ5 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the5^(th) position from the carboxyl end of a fatty acid substrate.Examples of Δ5 desaturases are listed in Table 1. In one embodiment, theΔ5 desaturase comprises amino acids having a sequence as provided in SEQID NO:26, a biologically active fragment thereof, or an amino acidsequence which is at least 80% identical to SEQ ID NO:26. In anotherembodiment, the Δ5 desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:13, a biologically active fragment thereof, or anamino acid sequence which is at least 53% identical to SEQ ID NO:13.

As used herein, a “Δ6 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the6^(th) position from the carboxyl end of a fatty acid substrate.Examples of Δ6 desaturases are listed in Table 1.

In one embodiment, the Δ6 desaturase is further characterised by havingat least two, preferably all three and preferably in a plant cell, ofthe following: i) greater Δ6 desaturase activity on α-linolenic acid(ALA, 18:3Δ9,12,15, ω3) than linoleic acid (LA, 18:2Δ9,12, ω6) as fattyacid substrate; ii) greater Δ6 desaturase activity on ALA-CoA as fattyacid substrate than on ALA joined to the sn-2 position of PC as fattyacid substrate; and iii) Δ8 desaturase activity on ETrA.

In another embodiment the Δ6 desaturase has greater activity on an ω3substrate than the corresponding ω6 substrate and has activity on ALA toproduce octadecatetraenoic acid (stearidonic acid, SDA, 18:4Δ6,9,12, 15,ω3) with an efficiency of at least 5%, more preferably at least 7.5%, ormost preferably at least 10% when expressed from an exogenouspolynucleotide in a recombinant cell, or at least 35% when expressed ina yeast cell. In one embodiment, the Δ6 desaturase has greater activity,for example, at least about a 2-fold greater Δ6 desaturase activity, onALA than LA as fatty acid substrate. In another embodiment, the Δ6desaturase has greater activity, for example, at least about 5 foldgreater Δ6 desaturase activity or at least 10-fold greater activity, onALA-CoA as fatty acid substrate than on ALA joined to the sn-2 positionof PC as fatty acid substrate.

In one embodiment, the Δ6 desaturase has no detectable Δ5 desaturaseactivity on ETA. In another embodiment, the Δ6 desaturase comprisesamino acids having a sequence as provided in SEQ ID NO:10, abiologically active fragment thereof, or an amino acid sequence which isat least 77% identical to SEQ ID NO:10. In another embodiment, the Δ6desaturase comprises amino acids having a sequence as provided in SEQ IDNO:8, a biologically active fragment thereof, or an amino acid sequencewhich is at least 67% identical to SEQ ID NO:8. The Δ6 desaturase mayalso have Δ8 desaturase activity.

As used herein, a “Δ8 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the8^(th) position from the carboxyl end of a fatty acid substrate. The Δ8desaturase is at least capable of converting ETrA to ETA. Examples of Δ8desaturases are listed in Table 1. In one embodiment, the Δ8 desaturasecomprises amino acids having a sequence as provided in SEQ ID NO:24, abiologically active fragment thereof, or an amino acid sequence which isat least 80% identical to SEQ ID NO:24.

As used herein, an “ω3 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the3rd position from the methyl end of a fatty acid substrate. Examples ofω3 desaturases include those described by Pereira et al. (2004a),Horiguchi et al. (1998), Berberich et al. (1998) and Spychalla et al.(1997).

In one embodiment, the ω3 desaturase is at least capable of convertingone of ARA to EPA, dGLA to ETA, γ-linolenic acid (GLA, 18:3Δ6,9,12, ω6)to SDA, both ARA to EPA and dGLA to ETA, both ARA to EPA and GLA to SDA,or all three of these.

In one embodiment, the ω3 desaturase has Δ17 desaturase activity on aC20 fatty acid which has at least three carbon-carbon double bonds,preferably ARA. In another embodiment, the ω3 desaturase has Δ15desaturase activity on a C18 fatty acid which has three carbon-carbondouble bonds, preferably GLA.

As used herein, a “Δ15 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the15^(th) position from the carboxyl end of a fatty acid substrate.

As used herein, a “Δ17 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the17^(th) position from the carboxyl end of a fatty acid substrate.

In another embodiment, the ω3 desaturase has greater activity on anacyl-CoA substrate, for example, ARA-CoA, than a corresponding acyl-PCsubstrate. As used herein, a “corresponding acyl-PC substrate” refers tothe fatty acid esterified at the sn-2 position of phosphatidylcholine(PC) where the fatty acid is the same fatty acid as in the acyl-CoAsubstrate. In an embodiment, the activity is at least two-fold greater.

In a further embodiment, the ω3 desaturase comprises amino acids havinga sequence as provided in SEQ ID NO:15, 17 or 20, a biologically activefragment thereof, or an amino acid sequence which is at least 35%identical to SEQ ID NO:15, at least 60% identical to SEQ ID NO:17 and/orat least 60% identical to SEQ ID NO:20.

In yet a further embodiment, a desaturase for use in the presentinvention has greater activity on an acyl-CoA substrate than acorresponding acyl-PC substrate. As outlined above, a “correspondingacyl-PC substrate” refers to the fatty acid esterified at the sn-2position of phosphatidylcholine (PC) where the fatty acid is the samefatty acid as in the acyl-CoA substrate. In an embodiment, the activityis at least two-fold greater. In an embodiment, the desaturase is a Δ5or Δ6 desaturase, examples of which are provided, but not limited to,those listed in Table 2.

TABLE 2 Desaturases with greater activity on an acyl-CoA substrate thana corresponding acyl-PC substrate Type of Accession Protein Enzymeorganism Species Nos. size (aa's) References Δ6-desaturase AlgaeMantoniella CAQ30479 449 Hoffmann et al. 2008 squamata OstreococcusAAW70159 456 Domergue et al. 2005 tauri Δ5-desaturase Algae MantoniellaCAQ30478 482 Hoffmann et al. 2008 squamata Plant Anemone N/A Sayanova etal. 2007 leveilleiElongases

Biochemical evidence suggests that the fatty acid elongation consists of4 steps: condensation, reduction, dehydration and a second reduction. Inthe context of this invention, an “elongase” refers to the polypeptidethat catalyses the condensing step in the presence of the other membersof the elongation complex, under suitable physiological conditions. Ithas been shown that heterologous or homologous expression in a cell ofonly the condensing component (“elongase”) of the elongation proteincomplex is required for the elongation of the respective acyl chain.Thus, the introduced elongase is able to successfully recruit thereduction and dehydration activities from the transgenic host to carryout successful acyl elongations. The specificity of the elongationreaction with respect to chain length and the degree of desaturation offatty acid substrates is thought to reside in the condensing component.This component is also thought to be rate limiting in the elongationreaction.

As used herein, a “Δ5 elongase” is at least capable of converting EPA toDPA. Examples of Δ5 elongases include those disclosed in WO2005/103253.In one embodiment, the Δ5 elongase has activity on EPA to produce DPAwith an efficiency of at least 60%, more preferably at least 65%, morepreferably at least 70% or most preferably at least 75%. In a furtherembodiment, the Δ5 elongase comprises an amino acid sequence as providedin SEQ ID NO:6, a biologically active fragment thereof, or an amino acidsequence which is at least 47% identical to SEQ ID NO:6

As used herein, a “Δ6 elongase” is at least capable of converting SDA toETA. Examples of Δ6 elongases include those listed in Table 1. In oneembodiment, the elongase comprises amino acids having a sequence asprovided in SEQ ID NO:4, a biologically active fragment thereof, or anamino acid sequence which is at least 55% identical to SEQ ID NO:4.

As used herein, a “Δ9 elongase” is at least capable of converting ALA toETrA. Examples of Δ9 elongases include those listed in Table 1. In oneembodiment, the Δ9 elongase comprises amino acids having a sequence asprovided in SEQ ID NO:22, a biologically active fragment thereof, or anamino acid sequence which is at least 80% identical to SEQ ID NO:22. Inanother embodiment, the Δ9 elongase comprises amino acids having asequence as provided in SEQ ID NO:28, a biologically active fragmentthereof, or an amino acid sequence which is at least 81% identical toSEQ ID NO:28. In another embodiment, the Δ9 elongase comprises aminoacids having a sequence as provided in SEQ ID NO:94, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 50%identical to SEQ ID NO:94. In another embodiment, the Δ9 elongasecomprises amino acids having a sequence as provided in SEQ ID NO:96, abiologically active fragment thereof, or an amino acid sequence which isat least 50% identical to SEQ ID NO:96. In a further embodiment, the Δ9elongase comprises amino acids having a sequence as provided in SEQ IDNO:94 or SEQ ID NO:96, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:94and/or SEQ ID NO:96, and wherein the elongase has greater activity on anω6 substrate than the corresponding ω3 substrate.

As used herein, the term “has greater activity on an ω6 substrate thanthe corresponding ω3 substrate” refers to the relative activity on theenzyme on substrates that differ by the action of an ω3 desaturase.Preferably, the ω6 substrate is LA and the ω3 substrate is ALA.

As used herein, an “elongase with Δ6 elongase and Δ9 elongase activity”is at least capable of (i) converting SDA to ETA and (ii) converting ALAto ETrA and has greater Δ6 elongase activity than Δ9 elongase activity.In one embodiment, the elongase has an efficiency of conversion on SDAto produce ETA which is at least 50%, more preferably at least 60%,and/or an efficiency of conversion on ALA to produce ETrA which is atleast 6% or more preferably at least 9%. In another embodiment, theelongase has at least about 6.5 fold greater Δ6 elongase activity thanΔ9 elongase activity. In a further embodiment, the elongase has nodetectable Δ5 elongase activity. In yet a further embodiment, theelongase comprises amino acids having a sequence as provided in SEQ IDNO:4, a biologically active fragment thereof, or an amino acid sequencewhich is at least 55% identical to SEQ ID NO:4.

Other Enzymes

As used herein, the term “diacylglycerol acyltransferase” (EC 2.3.1.20;DGAT) refers to a protein which transfers a fatty acyl group fromacyl-CoA to a diacylglycerol substrate to produce a triacylglycerol.Thus, the term “diacylglycerol acyltransferase activity” refers to thetransfer of acyl-CoA to diacylglycerol to produce triacylglycerol. Thereare three known types of DGAT referred to as DGAT1, DGAT2 and DGAT3respectively. DGAT1 polypeptides typically have 10 transmembranedomains, DGAT2 typically have 2 transmembrane domains, whilst DGAT3 istypically soluble. Examples of DGAT1 polypeptides include polypeptidesencoded by DGAT1 genes from Aspergillus fumigatus (Accession No.XP_755172), Arabidopsis thaliana (CAB44774), Ricinus communis(AAR11479), Vernicia fordii (ABC94472), Vernonia galamensis (ABV21945,ABV21946), Euonymus alatus (AAV31083), Caenorhabditis elegans(AAF82410), Rattus norvegicus (NP_445889), Homo sapiens (NP 036211), aswell as variants and/or mutants thereof. Examples of DGAT2 polypeptidesinclude polypeptides encoded by DGAT2 genes from Arabidopsis thaliana(Accession No. NP_566952), Ricinus communis (AAY16324), Vernicia fordii(ABC94474), Mortierella ramanniana (AAK84179), Homo sapiens (Q96PD7,Q58HT5), Bos taurus (Q70VD8), Mus musculus (AAK84175), MicromonasCCMP1545, as well as variants and/or mutants thereof. Examples of DGAT3polypeptides include polypeptides encoded by DGAT3 genes from peanut(Arachis hypogaea, Saha, et al., 2006), as well as variants and/ormutants thereof.

Polypeptides/Peptides

The invention also provides for polypeptides which may be purified orrecombinant. By “substantially purified polypeptide” or “purifiedpolypeptide” we mean a polypeptide that has generally been separatedfrom the lipids, nucleic acids, other peptides, and other contaminatingmolecules with which it is associated in a cell in which it is producedor in its native state. Preferably, the substantially purifiedpolypeptide is at least 60% free, more preferably at least 75% free, andmore preferably at least 90% free from other components in the cell inwhich it is produced or with which it is naturally associated.

The term “recombinant” in the context of a polypeptide refers to thepolypeptide when produced by a cell, or in a cell-free expressionsystem, in an altered amount or at an altered rate, compared to itsnative state if it is produced naturally. In one embodiment the cell isa cell that does not naturally produce the polypeptide. However, thecell may be a cell which comprises a non-endogenous gene that causes analtered amount of the polypeptide to be produced. A recombinantpolypeptide of the invention includes polypeptides in the cell, tissue,organ or organism, or cell-free expression system, in which it isproduced i.e. a polypeptide which has not been purified or separatedfrom other components of the transgenic (recombinant) cell in which itwas produced, and polypeptides produced in such cells or cell-freesystems which are subsequently purified away from at least some othercomponents.

The terms “polypeptide” and “protein” are generally usedinterchangeably.

A polypeptide or class of polypeptides may be defined by the extent ofidentity (% identity) of its amino acid sequence to a reference aminoacid sequence, or by having a greater % identity to one reference aminoacid sequence than to another. The % identity of a polypeptide to areference amino acid sequence is typically determined by GAP analysis(Needleman and Wunsch, 1970; GCG program) with parameters of a gapcreation penalty=5, and a gap extension penalty=0.3. The query sequenceis at least 15 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 15 amino acids. More preferably,the query sequence is at least 50 amino acids in length, and the GAPanalysis aligns the two sequences over a region of at least 50 aminoacids. More preferably, the query sequence is at least 100 amino acidsin length and the GAP analysis aligns the two sequences over a region ofat least 100 amino acids. Even more preferably, the query sequence is atleast 250 amino acids in length and the GAP analysis aligns the twosequences over a region of at least 250 amino acids. Even morepreferably, the GAP analysis aligns two sequences over their entirelength. The polypeptide or class of polypeptides may have the sameenzymatic activity as, or a different activity than, or lack theactivity of, the reference polypeptide. Preferably, the polypeptide hasan enzymatic activity of at least 10% of the activity of the referencepolypeptide.

As used herein a “biologically active” fragment is a portion of apolypeptide of the invention which maintains a defined activity of afull-length reference polypeptide, for example possessing desaturaseand/or elongase activity or other enzyme activity. Biologically activefragments as used herein exclude the full-length polypeptide.Biologically active fragments can be any size portion as long as theymaintain the defined activity. Preferably, the biologically activefragment maintains at least 10% of the activity of the full lengthprotein.

With regard to a defined polypeptide or enzyme, it will be appreciatedthat % identity figures higher than those provided herein will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide/enzyme comprisesan amino acid sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

In an embodiment, the substantially purified and/or recombinant Δ6desaturase of the invention does not comprise a sequence provided inaccession no. EEH58637.1 or XP_001421073.1. In another embodiment, thesubstantially purified and/or recombinant ω3 desaturase of the inventiondoes not comprise a sequence provided in accession no. XP_002505536.1.In another embodiment, the substantially purified and/or recombinantDGAT of the invention does not comprise a sequence provided in accessionno. EEH54819.1.

Amino acid sequence mutants of the polypeptides of the defined hereincan be prepared by introducing appropriate nucleotide changes into anucleic acid defined herein, or by in vitro synthesis of the desiredpolypeptide. Such mutants include, for example, deletions, insertions orsubstitutions of residues within the amino acid sequence. A combinationof deletion, insertion and substitution can be made to arrive at thefinal construct, provided that the final peptide product possesses thedesired characteristics.

Mutant (altered) peptides can be prepared using any technique known inthe art. For example, a polynucleotide of the invention can be subjectedto in vitro mutagenesis. Such in vitro mutagenesis techniques includesub-cloning the polynucleotide into a suitable vector, transforming thevector into a “mutator” strain such as the E. coli XL-1 red (Stratagene)and propagating the transformed bacteria for a suitable number ofgenerations. In another example, the polynucleotides of the inventionare subjected to DNA shuffling techniques as broadly described byHarayama (1998). Products derived from mutated/altered DNA can readilybe screened using techniques described herein to determine if theypossess desaturase and/or elongase activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active site(s). Other sites of interestare those in which particular residues obtained from various strains orspecies are identical. These positions may be important for biologicalactivity. These sites, especially those falling within a sequence of atleast three other identically conserved sites, are preferablysubstituted in a relatively conservative manner. Such conservativesubstitutions are shown in Table 3 under the heading of “exemplarysubstitutions”.

In a preferred embodiment a mutant/variant polypeptide has only, or notmore than, one or two or three or four conservative amino acid changeswhen compared to a naturally occurring polypeptide. Details ofconservative amino acid changes are provided in Table 3. As the skilledperson would be aware, such minor changes can reasonably be predictednot to alter the activity of the polypeptide when expressed in arecombinant cell.

TABLE 3 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

Also included within the scope of the invention are polypeptides definedherein which are differentially modified during or after synthesis, forexample, by biotinylation, benzylation, glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. These modifications may serve to increase thestability and/or bioactivity of the polypeptide of the invention.

Polypeptides can be produced in a variety of ways, including productionand recovery of natural polypeptides, production and recovery ofrecombinant polypeptides, and chemical synthesis of the polypeptides. Inone embodiment, a recombinant polypeptide is produced by culturing acell capable of expressing the polypeptide under conditions effective toproduce the polypeptide. The recombinant polypeptide may subsequently besecreted from the cell and recovered, or extracted from the cell andrecovered, and is preferably purified away from contaminating molecules.It may or may not be further modified chemically or enzymatically. Apreferred cell to culture is a recombinant cell defined herein.Effective culture conditions include, but are not limited to, effectivemedia, biorcactor, temperature, pH and oxygen conditions that permitpolypeptide production. An effective medium refers to any medium inwhich a cell is cultured to produce a polypeptide defined herein. Suchmedium typically comprises an aqueous medium having assimilable carbon,nitrogen and phosphate sources, and appropriate salts, minerals, metalsand other nutrients, such as vitamins. Cells defined herein can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes, and petri plates. Culturing can be carried outat a temperature, pH and oxygen content appropriate for a recombinantcell. Such culturing conditions are within the expertise of one ofordinary skill in the art. A more preferred cell to produce thepolypeptide is a cell in a plant, especially in a seed in a plant.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target analyte, as well as compoundscomprising said fragments. Such fragments include Fv, F(ab′) and F(ab′)₂fragments, as well as single chain antibodies (scFv). Antibodies of theinvention may be monoclonal or polyclonal and can be produced usingstandard procedures in the art.

Polynucleotides

The invention also provides for polynucleotides which may be, forexample, a gene, an isolated polynucleotide, or a chimeric DNA. It maybe DNA or RNA of genomic or synthetic origin, double-stranded orsingle-stranded, and combined with carbohydrate, lipids, protein orother materials to perform a particular activity defined herein. Theterm “polynucleotide” is used interchangeably herein with the term“nucleic acid molecule”. By “isolated polynucleotide” we mean apolynucleotide which, if obtained from a natural source, has beenseparated from the polynucleotide sequences with which it is associatedor linked in its native state, or a non-naturally occurringpolynucleotide. Preferably, the isolated polynucleotide is at least 60%free, more preferably at least 75% free, and more preferably at least90% free from other components with which it is naturally associated.

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising thetranscribed region and, if translated, the protein coding region, of astructural gene and including sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of at least about 2 kbon either end and which are involved in expression of the gene. In thisregard, the gene includes control signals such as promoters, enhancers,termination and/or polyadenylation signals that are naturally associatedwith a given gene, or heterologous control signals in which case thegene is referred to as a “chimeric gene”. The sequences which arelocated 5′ of the protein coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the protein coding region and which arepresent on the mRNA are referred to as 3′ non-translated sequences. Theterm “gene” encompasses both cDNA and genomic forms of a gene. A genomicform or clone of a gene contains the coding region which may beinterrupted with non-coding sequences termed “introns” or “interveningregions” or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (hnRNA). Introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns therefore areabsent in the messenger RNA (mRNA) transcript. The mRNA functions duringtranslation to specify the sequence or order of amino acids in a nascentpolypeptide. The term “gene” includes a synthetic or fusion moleculeencoding all or part of the proteins of the invention described hereinand a complementary nucleotide sequence to any one of the above.

As used herein, a “chimeric DNA” refers to any DNA molecule that is nota native DNA molecule in its native location, also referred to herein asa “DNA construct”. Typically, a chimeric DNA or chimeric gene comprisesregulatory and transcribed or protein coding sequences that are notfound together in nature. Accordingly, a chimeric DNA or chimeric genemay comprise regulatory sequences and coding sequences that are derivedfrom different sources, or regulatory sequences and coding sequencesderived from the same source, but arranged in a manner different thanthat found in nature.

The term “endogenous” is used herein to refer to a substance that isnormally present or produced in an unmodified plant at the samedevelopmental stage as the plant under investigation. An “endogenousgene” refers to a native gene in its natural location in the genome ofan organism. As used herein, “recombinant nucleic acid molecule”,“recombinant polynucleotide” or variations thereof refer to a nucleicacid molecule which has been constructed or modified by recombinant DNAtechnology. The terms “foreign polynucleotide” or “exogenouspolynucleotide” or “heterologous polynucleotide” and the like refer toany nucleic acid which is introduced into the genome of a cell byexperimental manipulations. Foreign or exogenous genes may be genes thatare inserted into a non-native organism, native genes introduced into anew location within the native host, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure. The terms “genetically modified”, “transgenic” and variationsthereof include introducing genes into cells by transformation ortransduction, mutating genes in cells and altering or modulating theregulation of a gene in a cell or organisms to which these acts havebeen done or their progeny. A “genomic region” as used herein refers toa position within the genome where a transgene, or group of transgenes(also referred to herein as a cluster), have been inserted into a cell,or an ancestor thereof. Such regions only comprise nucleotides that havebeen incorporated by the intervention of man such as by methodsdescribed herein.

The term “exogenous” in the context of a polynucleotide refers to thepolynucleotide when present in a cell in an altered amount compared toits native state. In one embodiment, the cell is a cell that does notnaturally comprise the polynucleotide. However, the cell may be a cellwhich comprises a non-endogenous polynucleotide resulting in an alteredamount of production of the encoded polypeptide. An exogenouspolynucleotide of the invention includes polynucleotides which have notbeen separated from other components of the transgenic (recombinant)cell, or cell-free expression system, in which it is present, andpolynucleotides produced in such cells or cell-free systems which aresubsequently purified away from at least some other components. Theexogenous polynucleotide (nucleic acid) can be a contiguous stretch ofnucleotides existing in nature, or comprise two or more contiguousstretches of nucleotides from different sources (naturally occurringand/or synthetic) joined to form a single polynucleotide. Typically suchchimeric polynucleotides comprise at least an open reading frameencoding a polypeptide of the invention operably linked to a promotersuitable of driving transcription of the open reading frame in a cell ofinterest.

As used herein, the term “different exogenous polynucleotides” orvariations thereof means that the nucleotide sequence of eachpolynucleotide are different by at least one, preferably more,nucleotides. The polynucleotides encode RNAs which may or may not betranslated to a protein within the cell. In an example, it is preferredthat each polynucleotide encodes a protein with a different activity. Inanother example, each exogenous polynucleotide is less than 95%, lessthan 90%, or less than 80% identical to the other exogenouspolynucleotides. Preferably, the exogenous polynucleotides encodefunctional proteins/enzymes. Furthermore, it is preferred that thedifferent exogenous polynucleotides are non-overlapping in that eachpolynucleotide is a distinct region of the, for example,extrachromosomal transfer nucleic acid which does not overlap withanother exogenous polynucleotide. At a minimum, each exogenouspolynucleotide has a transcription start and stop site, as well as thedesignated promoter. An individual exogenous polynucleotide may or maynot comprise introns.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

In an embodiment, the isolated and/or exogenous polynucleotide encodinga Δ6 desaturase of the invention does not comprise the sequence from theMicromonas or Ostreococcus genome predicted to encode the amino acidsequence provided in accession no. EEH58637.1 or XP_001421073.1respectively. In another embodiment, the isolated and/or exogenouspolynucleotide encoding a ω3 desaturase of the invention does notcomprise the sequence from the Micromonas genome predicted to encode theamino acid sequence provided in accession no. XP_002505536.1. In anotherembodiment, the isolated and/or exogenous polynucleotide encoding a DGATof the invention does not comprise the sequence from the Micromonasgenome predicted to encode the amino acid sequence provided in accessionno. EEH54819.1.

A polynucleotide of the present invention may selectively hybridise,under stringent conditions, to a polynucleotide that encodes apolypeptide of the present invention. As used herein, stringentconditions are those that (1) employ during hybridisation a denaturingagent such as formamide, for example, 50% (v/v) formamide with 0.1%(w/v) bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (2) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1%SDS and/or (3) employ low ionic strength and high temperature forwashing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at50° C.

Polynucleotides of the invention may possess, when compared to naturallyoccurring molecules, one or more mutations which are deletions,insertions, or substitutions of nucleotide residues. Polynucleotideswhich have mutations relative to a reference sequence can be eithernaturally occurring (that is to say, isolated from a natural source) orsynthetic (for example, by performing site-directed mutagenesis or DNAshuffling on the nucleic acid as described above). It is thus apparentthat polynucleotides of the invention can be either from a naturallyoccurring source or recombinant.

Recombinant Vectors

One embodiment of the present invention includes a recombinant vector,which comprises at least one polynucleotide molecule defined herein,inserted into any vector capable of delivering the polynucleotidemolecule into a host cell. Recombinant vectors include expressionvectors. Recombinant vectors contain heterologous polynucleotidesequences, that is polynucleotide sequences that are not naturally foundadjacent to polynucleotide molecules defined herein that preferably arederived from a species other than the species from which thepolynucleotide molecule(s) are derived. The vector can be either RNA orDNA, either prokaryotic or eukaryotic, and typically is a viral vector,derived from a virus, or a plasmid. Plasmid vectors typically includeadditional nucleic acid sequences that provide for easy selection,amplification, and transformation of the expression cassette inprokaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, orbinary vectors containing one or more T-DNA regions. Additional nucleicacid sequences include origins of replication to provide for autonomousreplication of the vector, selectable marker genes, preferably encodingantibiotic or herbicide resistance, unique multiple cloning sitesproviding for multiple sites to insert nucleic acid sequences or genesencoded in the nucleic acid construct, and sequences that enhancetransformation of prokaryotic and eukaryotic (especially plant) cells.The recombinant vector may comprise more than one polynucleotide definedherein, for example three, four, five or six polynucleotides of theinvention in combination, each operably linked to expression controlsequences that are operable in the cell of interest. Such more than onepolynucleotide of the invention, for example 3, 4, 5 or 6polynucleotides, are preferably covalently joined together in a singlerecombinant vector, which may then be introduced as a single moleculeinto a cell to form a recombinant cell according to the invention, andpreferably integrated into the genome of the recombinant cell, forexample in a transgenic plant. Thereby, the polynucleotides which are sojoined will be inherited together as a single genetic locus in progenyof the recombinant cell or plant. The recombinant vector or plant maycomprise two or more such recombinant vectors, each containing multiplepolynucleotides, for example wherein each recombinant vector comprises3, 4, 5 or 6 polynucleotides.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence, such as a polynucleotide definedherein, if it stimulates or modulates the transcription of the codingsequence in an appropriate cell. Generally, promoter transcriptionalregulatory elements that are operably linked to a transcribed sequenceare physically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

When there are multiple promoters present, each promoter mayindependently be the same or different.

Recombinant molecules such as the chimeric DNAs may also contain (a) oneor more secretory signals which encode signal peptide sequences, toenable an expressed polypeptide defined herein to be secreted from thecell that produces the polypeptide or which provide for localisation ofthe expressed polypeptide, for example for retention of the polypeptidein the endoplasmic reticulum (ER) in the cell or transfer into aplastid, and/or (b) contain fusion sequences which lead to theexpression of nucleic acid molecules as fusion proteins. Examples ofsuitable signal segments include any signal segment capable of directingthe secretion or localisation of a polypeptide defined herein. Preferredsignal segments include, but are not limited to, Nicotiana nectarinsignal peptide (U.S. Pat. No. 5,939,288), tobacco extensin signal or thesoy oleosin oil body binding protein signal. Recombinant molecules mayalso include intervening and/or untranslated sequences surroundingand/or within the nucleic acid sequences of nucleic acid moleculesdefined herein.

To facilitate identification of transformants, the nucleic acidconstruct desirably comprises a selectable or screenable marker gene as,or in addition to, the foreign or exogenous polynucleotide. By “markergene” is meant a gene that imparts a distinct phenotype to cellsexpressing the marker gene and thus allows such transformed cells to bedistinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, i.e., by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). The marker gene and the nucleotide sequence ofinterest do not have to be linked. The actual choice of a marker is notcrucial as long as it is functional (i.e., selective) in combinationwith the cells of choice such as a plant cell. The marker gene and theforeign or exogenous polynucleotide of interest do not have to belinked, since co-transformation of unlinked genes as, for example,described in U.S. Pat. No. 4,399,216 is also an efficient process inplant transformation.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, erythromycin, chloramphenicolor tetracycline resistance, preferably kanamycin resistance. Exemplaryselectable markers for selection of plant transformants include, but arenot limited to, a hyg gene which encodes hygromycin B resistance; aneomycin phosphotransferase (nptII) gene conferring resistance tokanamycin, paromomycin, G418; a glutathione-S-transferase gene from ratliver conferring resistance to glutathione derived herbicides as, forexample, described in EP 256223; a glutamine synthetase gene conferring,upon overexpression, resistance to glutamine synthetase inhibitors suchas phosphinothricin as, for example, described in WO 87/05327, anacetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as,for example, described by Hinchee et al. (1988), a bar gene conferringresistance against bialaphos as, for example, described in WO91/02071; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a dihydrofolatereductase (DHFR) gene conferring resistance to methotrexate (Thillet etal., 1988); a mutant acetolactate synthase gene (ALS), which confersresistance to imidazolinone, sulfonylurea or other ALS-inhibitingchemicals (EP 154,204); a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan; or a dalapon dehalogenasegene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known, a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known, an aequorin gene(Prasher et al., 1985), which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (Niedz etal., 1995) or derivatives thereof; a luciferase (luc) gene (Ow et al.,1986), which allows for bioluminescence detection, and others known inthe art. By “reporter molecule” as used in the present specification ismeant a molecule that, by its chemical nature, provides an analyticallyidentifiable signal that facilitates determination of promoter activityby reference to protein product.

Preferably, the nucleic acid construct is stably incorporated into thegenome of the cell, such as the plant cell. Accordingly, the nucleicacid may comprise appropriate elements which allow the molecule to beincorporated into the genome, or the construct is placed in anappropriate vector which can be incorporated into a chromosome of thecell.

Expression

As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of oneor more specified polynucleotide molecule(s). Preferably, the expressionvector is also capable of replicating within the host cell. Expressionvectors can be either prokaryotic or eukaryotic, and are typicallyviruses or plasmids. Expression vectors of the present invention includeany vectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal,endoparasite, arthropod, animal, and plant cells. Particularly preferredexpression vectors of the present invention can direct gene expressionin yeast and/or plant cells.

Expression vectors of the present invention contain regulatory sequencessuch as transcription control sequences, translation control sequences,origins of replication, and other regulatory sequences that arecompatible with the recombinant cell and that control the expression ofpolynucleotide molecules of the present invention. In particular,polynucleotides or vectors of the present invention includetranscription control sequences. Transcription control sequences aresequences which control the initiation, elongation, and termination oftranscription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. The choice of the regulatory sequences used depends on thetarget organism such as a plant and/or target organ or tissue ofinterest. Such regulatory sequences may be obtained from any eukaryoticorganism such as plants or plant viruses, or may be chemicallysynthesized. A variety of such transcription control sequences are knownto those skilled in the art. Particularly preferred transcriptioncontrol sequences are promoters active in directing transcription inplants, either constitutively or stage and/or tissue specific, dependingon the use of the plant or parts thereof.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliformvirus promoter, the commelina yellow mottle virus promoter, thelight-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase, the rice cytosolictriosephosphate isomerase promoter, the adeninephosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 genepromoter, the mannopine synthase and octopine synthase promoters, theAdh promoter, the sucrose synthase promoter, the R gene complexpromoter, and the chlorophyll α/β binding protein gene promoter. Thesepromoters have been used to create DNA vectors that have been expressedin plants; see, e.g., WO 84/02913. All of these promoters have been usedto create various types of plant-expressible recombinant DNA vectors.

For the purpose of expression in source tissues of the plant, such asthe leaf, seed, root or stem, it is preferred that the promotersutilized in the present invention have relatively high expression inthese specific tissues. For this purpose, one may choose from a numberof promoters for genes with tissue- or cell-specific or -enhancedexpression. Examples of such promoters reported in the literatureinclude the chloroplast glutamine synthetase GS2 promoter from pea, thechloroplast fructose-1,6-biphosphatase promoter from wheat, the nuclearphotosynthetic ST-LS1 promoter from potato, the serine/threonine kinasepromoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana.Also reported to be active in photosynthetically active tissues are theribulose-1,5-bisphosphate carboxylase promoter from eastern larch (Larixlaricina), the promoter for the Cab gene, Cab6, from pine, the promoterfor the Cab-1 gene from wheat, the promoter for the Cab-1 gene fromspinach, the promoter for the Cab 1R gene from rice, the pyruvate,orthophosphate dikinase (PPDK) promoter from Zea mays, the promoter forthe tobacco Lhcb1*2 gene, the Arabidopsis thaliana Suc2 sucrose-H³⁰symporter promoter, and the promoter for the thylakoid membrane proteingenes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).

Other promoters for the chlorophyll α/β-binding proteins may also beutilized in the present invention, such as the promoters for LhcB geneand PsbP gene from white mustard (Sinapis alba). A variety of plant genepromoters that are regulated in response to environmental, hormonal,chemical, and/or developmental signals, also can be used for expressionof RNA-binding protein genes in plant cells, including promotersregulated by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcSpromoter); (3) hormones, such as abscisic acid, (4) wounding (e.g.,WunI); or (5) chemicals, such as methyl jasmonate, salicylic acid,steroid hormones, alcohol, Safeners (WO 97/06269), or it may also beadvantageous to employ (6) organ-specific promoters.

As used herein, the term “plant storage organ specific promoter” refersto a promoter that preferentially, when compared to other plant tissues,directs gene transcription in a storage organ of a plant. Preferably,the promoter only directs expression of a gene of interest in thestorage organ, and/or expression of the gene of interest in other partsof the plant such as leaves is not detectable by Northern blot analysisand/or RT-PCR. Typically, the promoter drives expression of genes duringgrowth and development of the storage organ, in particular during thephase of synthesis and accumulation of storage compounds in the storageorgan. Such promoters may drive gene expression in the entire plantstorage organ or only part thereof such as the scedcoat, embryo orcotyledon(s) in seeds of dicotyledonous plants or the endosperm oraleurone layer of a seeds of monocotyledonous plants.

For the purpose of expression in sink tissues of the plant, such as thetuber of the potato plant, the fruit of tomato, or the seed of soybean,canola, cotton, Zea mays, wheat, rice, and barley, it is preferred thatthe promoters utilized in the present invention have relatively highexpression in these specific tissues. A number of promoters for geneswith tuber-specific or -enhanced expression are known, including theclass I patatin promoter, the promoter for the potato tuber ADPGPPgenes, both the large and small subunits, the sucrose synthase promoter,the promoter for the major tuber proteins including the 22 kD proteincomplexes and proteinase inhibitors, the promoter for the granule boundstarch synthase gene (GBSS), and other class I and II patatinspromoters. Other promoters can also be used to express a protein inspecific tissues, such as seeds or fruits. The promoter forβ-conglycinin or other seed-specific promoters such as the napin, zein,linin and phaseolin promoters, can be used. Root specific promoters mayalso be used. An example of such a promoter is the promoter for the acidchitinase gene. Expression in root tissue could also be accomplished byutilizing the root specific subdomains of the CaMV 35S promoter thathave been identified.

In a particularly preferred embodiment, the promoter directs expressionin tissues and organs in which fatty acid and oil biosynthesis takeplace. Such promoters act in seed development at a suitable time formodifying oil composition in seeds.

In a further particularly preferred embodiment, and in some aspects ofthe invention, the promoter is a plant storage organ specific promoter.In one embodiment, the plant storage organ specific promoter is a seedspecific promoter. In a more preferred embodiment, the promoterpreferentially directs expression in the cotyledons of a dicotyledonousplant or in the endosperm of a monocotyledonous plant, relative toexpression in the embryo of the seed or relative to other organs in theplant such as leaves. Preferred promoters for seed-specific expressioninclude i) promoters from genes encoding enzymes involved in fatty acidbiosynthesis and accumulation in seeds, such as desaturases andelongases, ii) promoters from genes encoding seed storage proteins, andiii) promoters from genes encoding enzymes involved in carbohydratebiosynthesis and accumulation in seeds. Seed specific promoters whichare suitable are the oilseed rape napin gene promoter (U.S. Pat. No.5,608,152), the Vida faba USP promoter (Baumlein et al., 1991), theArabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgarisphaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter(WO 91/13980) or the legumin B4 promoter (Baumlein et al., 1992), andpromoters which lead to the seed-specific expression in monocots such asmaize, barley, wheat, rye, rice and the like. Notable promoters whichare suitable are the barley 1pt2 or 1pt1 gene promoter (WO 95/15389 andWO 95/23230) or the promoters described in WO 99/16890 (promoters fromthe barley hordein gene, the rice glutelin gene, the rice oryzin gene,the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene,the maize zein gene, the oat glutelin gene, the sorghum kasirin gene,the rye secalin gene). Other promoters include those described by Brounet al. (1998), Potenza et al. (2004), US 20070192902 and US 20030159173.In an embodiment, the seed specific promoter is preferentially expressedin defined parts of the seed such as the cotyledon(s) or the endosperm.Examples of cotyledon specific promoters include, but are not limitedto, the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter(Perrin et al., 2000), and the bean phytohemagglutnin promoter (Perrinet al., 2000). Examples of endosperm specific promoters include, but arenot limited to, the maize zein-1 promoter (Chikwamba et al., 2003), therice glutelin-1 promoter (Yang et al., 2003), the barley D-hordeinpromoter (Horvath et al., 2000) and wheat HMW glutenin promoters(Alvarez et al., 2000). In a further embodiment, the seed specificpromoter is not expressed, or is only expressed at a low level, in theembryo and/or after the seed germinates.

In another embodiment, the plant storage organ specific promoter is atuber specific promoter. Examples include, but are not limited to, thepotato patatin B33, PAT21 and GBSS, promoters, as well as the sweetpotato sporamin promoter (for review see Potenza et al., 2004). In apreferred embodiment, the promoter directs expression preferentially inthe pith of the tuber, relative to the outer layers (skin, bark) or theembryo of the tuber.

In another embodiment, the plant storage organ specific promoter is afruit specific promoter. Examples include, but are not limited to, thetomato polygalacturonase, E8 and Pds promoters, as well as the apple ACCoxidase promoter (for review see Potenza et al., 2004). In a preferredembodiment, the promoter preferentially directs expression in the edibleparts of the fruit, for example the pith of the fruit, relative to theskin of the fruit or the seeds within the fruit.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the polynucleotideof the present invention, or may be heterologous with respect to thecoding region of the enzyme to be produced, and can be specificallymodified if desired so as to increase translation of mRNA. For a reviewof optimizing expression of transgenes, see Koziel et al. (1996). The 5′non-translated regions can also be obtained from plant viral RNAs(Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus,Alfalfa mosaic virus, among others) from suitable eukaryotic genes,plant genes (wheat and maize chlorophyll a/b binding protein geneleader), or from a synthetic gene sequence. The present invention is notlimited to constructs wherein the non-translated region is derived fromthe 5′ non-translated sequence that accompanies the promoter sequence.The leader sequence could also be derived from an unrelated promoter orcoding sequence. Leader sequences useful in context of the presentinvention comprise the maize Hsp70 leader (U.S. Pat. No. 5,362,865 andU.S. Pat. No. 5,859,347), and the TMV omega element as exemplified inExample 8.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the chimeric vector to thepolynucleotide of interest. The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ untranslated region, the 3′ untranslated region from pea smallsubunit Rubisco gene, the 3′ untranslated region from soybean 7S seedstorage protein gene are commonly used in this capacity. The 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide molecule by manipulating, for example, thenumber of copies of the polynucleotide molecule within a host cell, theefficiency with which those polynucleotide molecules are transcribed,the efficiency with which the resultant transcripts are translated, andthe efficiency of post-translational modifications. Recombinanttechniques useful for increasing the expression of polynucleotidemolecules defined herein include, but are not limited to, operativelylinking polynucleotide molecules to high-copy number plasmids,integration of the polynucleotide molecule into one or more host cellchromosomes, addition of vector stability sequences to plasmids,substitutions or modifications of transcription control signals (e.g.,promoters, operators, enhancers), substitutions or modifications oftranslational control signals (e.g., ribosome binding sites,Shine-Dalgarno sequences), modification of polynucleotide molecules tocorrespond to the codon usage of the host cell, and the deletion ofsequences that destabilize transcripts.

Transfer Nucleic Acids

Transfer nucleic acids of the invention at least comprise one,preferably two, border sequences and an exogenous polynucleotide. Thetransfer nucleic acid may or may not encode a selectable marker.Preferably, the transfer nucleic acid forms part of a binary vector inthe bacterium, where the binary vector further comprises elements whichallows replication of the vector in the bacterium or allows selection ormaintenance of bacterial cells containing the vector. Upon transfer to aeukaryotic cell the transfer nucleic acid component of the binary vectoris capable of integration into the genome of the eukaryotic cell.

As used herein, the term “extrachromosomal transfer nucleic acid” refersto a nucleic acid molecule that is capable of being transferred from abacterium, such as Agrobacterium sp., to a eukaryotic cell, such as aplant leaf cell. An extrachromosomal transfer nucleic acid is a geneticelement that is well-known as an element capable of being transferredwith the subsequent integration of a nucleotide sequence containedwithin its borders into the genome of the recipient cell. In thisrespect, a transfer nucleic acid is flanked, typically, by two “border”sequences, although in some instances a single border at one end can beused and the second end of the transferred nucleic acid is generatedrandomly in the transfer process. A desired exogenous polynucleotide istypically positioned between the left border-like sequence and the rightborder-like sequence of a transfer nucleic acid. The desiredpolynucleotide contained within the transfer nucleic acid may beoperably linked to a variety of different promoter and terminatorregulatory elements that facilitate its expression, i.e., transcriptionand/or translation of the polynucleotide. T-DNAs from Agrobacterium sp.such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, and manmade variants/mutants thereof are probably the best characterizedexamples of transfer nucleic acids. Another example is P-DNA(“plant-DNA”) which comprises T-DNA border-like sequences from plants.

As used herein, “T-DNA” refers to, for example, T-DNA of anAgrobacterium tumefaciens Ti plasmid or from an Agrobacterium rhizogenesRi plasmid, or a man made variants thereof which function as T-DNA(transferred-DNA). The T-DNA may comprise an entire T-DNA including bothright and left border sequences, but need only comprise the minimalsequences required in cis for transfer i.e., the right and T-DNA bordersequence. The T-DNAs of the invention have inserted into them, anywherebetween the right and left border sequences (if present), the exogenouspolynucleotide flanked by target sites for a site-specific recombinase.The sequences encoding factors required in trans for transfer of theT-DNA into a plant cell, such as vir genes, may be inserted into theT-DNA, or may be present on the same replicon as the T-DNA, orpreferably are in trans on a compatible replicon in the Agrobacteriumhost. Such “binary vector systems” are well known in the art.

As used herein, “P-DNA” refers to a transfer nucleic acid isolated froma plant genome, or a man made variants/mutants thereof, and comprises ateach end, or at only one end, a T-DNA border-like sequence. Theborder-like sequence preferably shares at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90% or at least 95%, butless than 100% sequence identity, with a T-DNA border sequence from anAgrobacterium sp., such as Agrobacterium tumefaciens or Agrobacteriumrhizogenes. Thus, P-DNAs can be used instead of T-DNAs to transfer anucleotide sequence contained within the P-DNA from, for exampleAgrobacterium, to another cell. The P-DNA, before insertion of theexogenous polynucleotide which is to be transferred, may be modified tofacilitate cloning and should preferably not encode any proteins. TheP-DNA is characterized in that it contains, at least a right bordersequence and preferably also a left border sequence.

As used herein, a “border” sequence(s) of a transfer nucleic acid can beisolated from selected organism such as a plant or bacteria, or be a manmade variant/mutant thereof. The border sequence promotes andfacilitates the transfer of the exogenous polynucleotide to which it islinked and may facilitate its integration in the recipient cell genome.

In an embodiment, a border-sequence is between 5-100 bp in length, 10-80bp in length, 15-75 bp in length, 15-60 bp in length, 15-50 bp inlength, 15-40 bp in length, 15-30 bp in length, 16-30 bp in length,20-30 bp in length, 21-30 bp in length, 22-30 bp in length, 23-30 bp inlength, 24-30 bp in length, 25-30 bp in length, or 26-30 bp in length.

Border sequences from T-DNA from Agrobacterium sp. are well known in theart and include those described in Lacroix et al. (2008), Tzfira andCitovsky (2006) and Glevin (2003). The border sequences of P-DNA can beisolated from any plant, such as from potato and wheat. In anembodiment, the P-DNA has the nucleic acid sequence ANGATNTATN6GT (SEQID NO:109), where “N” is any nucleotide, such as those represented by“A,” “G,” “C,” or “T”. Examples of other border sequences useful for theinvention include, but are not limited to,

(SEQ ID NO: 110) TGACAGGATATATTGGCGGGTAAAC; (SEQ ID NO: 111)TGGCAGGATATATTGTGGTGTAAAC; (SEQ ID NO: 112) TGGCAGGATATATACCGTTGTAATT;(SEQ ID NO: 113) CGGCAGGATATATTCAATTGTAATT; (SEQ ID NO: 114)TGGTAGGATATATACCGTTGTAATT; (SEQ ID NO: 115) TGGCAGGATATATGGTACTGTAATT;(SEQ ID NO: 116) YGRYAGGATATATWSNVBKGTAAWY; (SEQ ID NO: 117)CGGCAGGATATATCCTGATGTAAAT; (SEQ ID NO: 118) TGGCAGGAGTTATTCGAGGGTAAAC;(SEQ ID NO: 119) TGACAGGATATATCGTGATGTCAAC; (SEQ ID NO: 120)GGGAAGTACATATTGGCGGGTAAAC; (SEQ ID NO: 121) TTACAGGATATATTAATATGTATGA;(SEQ ID NO: 122) TAACATGATATATTCCCTTGTAAAT; (SEQ ID NO: 123)TGACAGGATATATGGTAATGTAAAC; and (SEQ ID NO: 124)TGGCAGGATATATACCGATGTAAAC, where * Y = C or T; R = A or G; K = G or T;W = A or T; S = C or G; V = A, C, or G; B = C, G, or T.

Whilst traditionally only Agrobacterium sp. have been used transfergenes to plants cells, there are now a large number of systems whichhave been identified/developed which act in a similar manner toAgrobacterium sp. Several non-Agrobacterium species have recently beengenetically modified to be competent for gene transfer (Chung et al.,2006; Broothaerts et al., 2005). These include Rhizobium sp. NGR234,Sinorhizobium meliloti and Mezorhizobium loti. The bacteria are madecompetent for gene transfer by providing the bacteria with the machineryneeded for the transformation process: i.e. a set of virulence genesencoded by an Agrobacterium Ti-plasmid and the T-DNA segment residing ona separate, small binary plasmid. Bacteria engineered in this way arecapable of transforming different plant tissues (leaf disks, calli andoval tissue), monocots or dicots, and various different plant species(eg. tobacco, rice).

Direct transfer of eukaryotic expression plasmids from bacteria toeukaryotic hosts was first achieved several decades ago by the fusion ofmammalian cells and protoplasts of plasmid-carrying Escherichia coli(Schaffner, 1980). Since then, the number of bacteria capable ofdelivering genes into mammalian cells has steadily increased (Weiss,2003), being discovered by four groups independently (Sizemore et al.1995; Courvalin et al., 1995; Powell et al., 1996; Darji et al., 1997).

Attenuated Shigella flexneri, Salmonella typhimurium or E. coli that hadbeen rendered invasive by the virulence plasmid (pWR100) of S. flexnerihave been shown to be able to transfer expression plasmids afterinvasion of host cells and intracellular death due to metabolicattenuation. Mucosal application, either nasally or orally, of suchrecombinant Shigella or Salmonella induced immune responses against theantigen that was encoded by the expression plasmids. In the meantime,the list of bacteria that was shown to be able to transfer expressionplasmids to mammalian host cells in vitro and in vivo has been more thandoubled and has been documented for S. typhi, S. choleraesuis, Listeriamonocytogenes, Yersinia pseudotuberculosis, and Y. enterocolitica(Fennelly et al., 1999; Shiau et al., 2001; Dietrich et al., 1998, 2001;Hense et al., 2001; Al-Mariri et al., 2002).

In general, it could be assumed that all bacteria that are able to enterthe cytosol of the host cell (like S. flexneri or L. monocytogenes) andlyse within this cellular compartment, should be able to transfer DNA.This is known as ‘abortive’ or ‘suicidal’ invasion as the bacteria haveto lyse for the DNA transfer to occur (Grillot-Courvalin et al., 1999).In addition, even many of the bacteria that remain in the phagocyticvacuole (like S. typhimurium) may also be able to do so. Thus,recombinant laboratory strains of E. coli that have been engineered tobe invasive but are unable of phagosomal escape, could deliver theirplasmid load to the nucleus of the infected mammalian cell nevertheless(Grillot-Courvalin et al., 1998). Furthermore, Agrobacterium tumefacienshas recently also been shown to introduce transgenes into mammaliancells (Kunik et al., 2001).

The transfer process using extrachromosomal transfer elements typicallytransfers multiple copies of the element into the recipient cell. Asused herein, the term “transiently transfected” means that although someof the exogenous polynucleotides may become stably integrated into thegenome of the cell, the cells are not selected for stable integration.As a result, much of the transfer nucleic acid remains extrachromosomalin the cell, for example greater than 90% of the copies of the exogenouspolynucleotide that are transferred into the recipient cell are notintegrated into the genome.

As used herein, the terms “transfection”, “transformation” andvariations thereof are generally used interchangeably. “Transfected” or“transformed” cells may have been manipulated to introduce the exogenouspolynucleotide(s), or may be progeny cells derived therefrom.

Recombinant Cells

The invention also provides a recombinant cell, preferably a recombinantplant cell, which is a host cell transformed with one or morerecombinant molecules, such as the polynucleotides, chimeric DNAs orrecombinant vectors defined herein. The recombinant cell may compriseany combination thereof, such as two or three recombinant vectors, or arecombinant vector and one or more additional polynucleotides orchimeric DNAs. Suitable cells of the invention include any cell that canbe transformed with a polynucleotide, chimeric DNA or recombinant vectorof the invention, such as for example, a molecule encoding a polypeptideor enzyme described herein. The cell is preferably a cell which isthereby capable of being used for producing LC-PUFA. The recombinantcell may be a cell in culture, a cell in vitro, or in an organism suchas for example a plant, or in an organ such as for example a seed or aleaf. Preferably, the cell is in a plant, more preferably in the seed ofa plant.

Host cells into which the polynucleotide(s) are introduced can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid molecule. Such nucleic acid molecules may be related toLC-PUFA synthesis, or unrelated. Host cells of the present inventioneither can be endogenously (i.e., naturally) capable of producingproteins defined herein, in which case the recombinant cell derivedtherefrom has an enhanced capability of producing the polypeptides, orcan be capable of producing such proteins only after being transformedwith at least one polynucleotide of the invention. In an embodiment, arecombinant cell of the invention has an enhanced capacity to synthesizea long chain polyunsaturated fatty acid. As used herein, the term “cellwith an enhanced capacity to synthesize a long chain polyunsaturatedfatty acid” is a relative term where the recombinant cell of theinvention is compared to the host cell lacking the polynucleotide(s) ofthe invention, with the recombinant cell producing more long chainpolyunsaturated fatty acids, or a greater concentration of LC-PUFA suchas EPA, DPA or DHA (relative to other fatty acids), than the nativecell. The cell with an enhanced capacity to synthesize another product,such as for example another fatty acid, a lipid, a carbohydrate such asstarch, an RNA molecule, a polypeptide, a pharmaceutical or otherproduct has a corresponding meaning.

Host cells of the present invention can be any cell capable of producingat least one protein described herein, and include bacterial, fungal(including yeast), parasite, arthropod, animal and plant cells. Thecells may be prokaryotic or eukaryotic. Preferred host cells are yeastand plant cells. In a preferred embodiment, the plant cell is a seedcell, in particular a cell in a cotyledon or endosperm of a seed. In oneembodiment, the cell is an animal cell or an algal cell. The animal cellmay be of any type of animal such as, for example, a non-human animalcell, a non-human vertebrate cell, a non-human mammalian cell, or cellsof aquatic animals such as, fish or crustacea, invertebrates, insects,etc. Non limiting examples of arthropod cells include insect cells suchas Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21, Trichoplusia nicells, and Drosophila S2 cells. An example of a bacterial cell useful asa host cell of the present invention is Synechococcus spp. (also knownas Synechocystis spp.), for example Synechococcus elongatus.

The cells may be of an organism suitable for a fermentation process. Asused herein, the term the “fermentation process” refers to anyfermentation process or any process comprising a fermentation step. Afermentation process includes, without limitation, fermentationprocesses used to produce alcohols (e.g., ethanol, methanol, butanol);organic acids (e.g., citric acid, acetic acid, itaconic acid, lacticacid, gluconic acid); ketones (e.g., acetone); amino acids (e.g.,glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillinand tetracycline); enzymes; vitamins (e.g., riboflavin, beta-carotene);and hormones. Fermentation processes also include fermentation processesused in the consumable alcohol industry (e.g., beer and wine), dairyindustry (e.g., fermented dairy products), leather industry and tobaccoindustry. Preferred fermentation processes include alcohol fermentationprocesses, as are well known in the art. Preferred fermentationprocesses are anaerobic fermentation processes, as are well known in theart. Suitable fermenting cells, typically microorganisms are able toferment, i.e., convert, sugars, such as glucose or maltose, directly orindirectly into the desired fermentation product. Examples of fermentingmicroorganisms include fungal organisms, such as yeast. As used herein,“yeast” includes Saccharomyces spp., Saccharomyces cerevisiae,Saccharomyces carlbergensis, Candida spp., Kluveromyces spp., Pichiaspp., Hansenula spp., Trichoderma spp., Lipomyces starkey, and Yarrowialipolytica. Preferred yeast include strains of the Saccharomyces spp.,and in particular, Saccharomyces cerevisiae.

Transgenic Plants

The invention also provides a plant comprising a cell of the invention,such as a transgenic plant comprising one or more polynucleotides of theinvention. The term “plant” as used herein as a noun refers to wholeplants, but as used as an adjective refers to any substance which ispresent in, obtained from, derived from, or related to a plant, such asfor example, plant organs (e.g. leaves, stems, roots, flowers), singlecells (e.g. pollen), seeds, plant cells and the like. The term “plantpart” refers to all plant parts that comprise the plant DNA, includingvegetative structures such as, for example, leaves or stems, roots,floral organs or structures, pollen, seed, seed parts such as an embryo,endosperm, scutellum or seed coat, plant tissue such as, for example,vascular tissue, cells and progeny of the same.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a gene construct (“transgene”) not foundin a wild-type plant of the same species, variety or cultivar.Transgenic plants as defined in the context of the present inventioninclude plants and their progeny which have been genetically modifiedusing recombinant techniques to cause production of at least onepolypeptide defined herein in the desired plant or plant organ.Transgenic plant cells and transgenic plant parts have correspondingmeanings. A “transgene” as referred to herein has the normal meaning inthe art of biotechnology and includes a genetic sequence which has beenproduced or altered by recombinant DNA or RNA technology and which hasbeen introduced into a cell of the invention, preferably a plant cell.The transgene may include genetic sequences derived from a plant cellwhich may be of the same species, variety or cultivar as the plant cellinto which the transgene is introduced or of a different species,variety or cultivar, or from a cell other than a plant cell. Typically,the transgene has been introduced into the cell, such as a plant, byhuman manipulation such as, for example, by transformation but anymethod can be used as one of skill in the art recognizes.

The terms “seed” and “grain” are used interchangeably herein. “Grain”refers to mature grain such as harvested grain or grain which is stillon a plant but ready for harvesting, but can also refer to grain afterimbibition or germination, according to the context. Mature graincommonly has a moisture content of less than about 18-20%. “Developingseed” as used herein refers to a seed prior to maturity, typically foundin the reproductive structures of the plant after fertilisation oranthesis, but can also refer to such seeds prior to maturity which areisolated from a plant.

As used herein, the term “plant storage organ” refers to a part of aplant specialized to storage energy in the form of, for example,proteins, carbohydrates, fatty acids and/or oils. Examples of plantstorage organs are seed, fruit, tuberous roots, and tubers. A preferredplant storage organ of the invention is seed.

As used herein, the term “phenotypically normal” refers to a geneticallymodified plant or plant organ, particularly a storage organ such as aseed, tuber or fruit of the invention not having a significantly reducedability to grow and reproduce when compared to an unmodified plant orplant organ. In an embodiment, the genetically modified plant or plantorgan which is phenotypically normal comprises an exogenouspolynucleotide encoding a silencing suppressor operably linked to aplant storage organ specific promoter and has an ability to grow orreproduce which is essentially the same as an isogenic plant or organnot comprising said polynucleotide. Preferably, the biomass, growthrate, germination rate, storage organ size, seed size and/or the numberof viable seeds produced is not less than 90% of that of a plant lackingsaid exogenous polynucleotide when grown under identical conditions.This term does not encompass features of the plant which may bedifferent to the wild-type plant but which do not affect the usefulnessof the plant for commercial purposes such as, for example, a ballerinaphenotype of seedling leaves.

Plants provided by or contemplated for use in the practice of thepresent invention include both monocotyledons and dicotyledons. Inpreferred embodiments, the plants of the present invention are cropplants (for example, cereals and pulses, maize, wheat, potatoes,tapioca, rice, sorghum, millet, cassava, barley, or pea), or otherlegumes. The plants may be grown for production of edible roots, tubers,leaves, stems, flowers or fruit. The plants may be vegetables orornamental plants. The plants of the invention may be: corn (Zea mays),canola (Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum),alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale),sorghum (Sorghum bicolour, Sorghum vulgare), sunflower (Helianthusannus), wheat (Tritium aestivum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachishypogaea), cotton (Gossypium hirsutum), sweet potato (Lopmoea batatus),cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocosnucifera), pineapple (Anana comosus), citrus tree (Citrus spp.), cocoa(Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado(Persea americana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew(Anacardium occidentale), macadamia (Macadamia intergrifolia), almond(Prunus amygdalus), sugar beets (Beta vulgaris), oats, or barley.

In a preferred embodiment, the plant is an angiosperm.

In an embodiment, the plant is an oilseed plant, preferably an oilseedcrop plant. As used herein, an “oilseed plant” is a plant species usedfor the commercial production of oils from the seeds of the plant. Theoilseed plant may be oil-seed rape (such as canola), maize, sunflower,soybean, sorghum, flax (linseed) or sugar beet. Furthermore, the oilseedplant may be other Brassicas, cotton, peanut, poppy, mustard, castorbean, sesame, safflower, or nut producing plants. The plant may producehigh levels of oil in its fruit, such as olive, oil palm or coconut.Horticultural plants to which the present invention may be applied arelettuce, endive, or vegetable brassicas including cabbage, broccoli, orcauliflower. The present invention may be applied in tobacco, cucurbits,carrot, strawberry, tomato, or pepper.

In a further preferred embodiment, the non-transgenic plant used toproduce a transgenic plant of the invention produces oil, especially inthe seed, which has i) less than 20%, less than 10% or less than 5% 18:2fatty acids and/or ii) less than 10% or less than 5% 18:3 fatty acids.

In a preferred embodiment, the transgenic plant is homozygous for eachand every gene that has been introduced (transgene) so that its progenydo not segregate for the desired phenotype. The transgenic plant mayalso be heterozygous for the introduced transgene(s), preferablyuniformly heterozygous for the transgene, such as for example in F1progeny which have been grown from hybrid seed. Such plants may provideadvantages such as hybrid vigour, well known in the art.

Where relevant, the transgenic plants may also comprise additionaltransgenes encoding enzymes involved in the production of LC-PUFAs suchas, but not limited to, a Δ6 desaturase, a Δ9 elongase, a Δ8 desaturase,a Δ6 elongase, a Δ5 desaturase, an ω3 desaturase, a Δ4 desaturase, a Δ5elongase, diacylglycerol acyltransferase, a Δ17 desaturase, a Δ15desaturase and/or a Δ12 desaturase. Examples of such enzymes with one ofmore of these activities are known in the art and include thosedescribed herein and in WO 05/103253 (see, for example, Table 1 of WO05/103253). In specific examples, the transgenic plant at leastcomprises exogenous polynucleotides encoding;

a) a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ5 elongase anda Δ6 elongase,

b) a Δ4 desaturase, a Δ5 desaturase, a Δ8 desaturase, a Δ5 elongase anda Δ9 elongase,

c) a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ5 elongase, aΔ6 elongase, and a Δ15 desaturase,

d) a Δ4 desaturase, a Δ5 desaturase, a Δ8 desaturase, a Δ5 elongase, aΔ9 elongase, and a Δ15 desaturase,

e) a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ5 elongase, aΔ6 elongase, and a Δ17 desaturase, or

f) a Δ4 desaturase, a Δ5 desaturase, a Δ8 desaturase, a Δ5 elongase, aΔ9 elongase, and a Δ17 desaturase.

Transformation of Plants

Transgenic plants can be produced using techniques known in the art,such as those generally described in A. Slater et al., PlantBiotechnology—The Genetic Manipulation of Plants, Oxford UniversityPress (2003), and P. Christou and H. Klee, Handbook of PlantBiotechnology, John Wiley and Sons (2004).

As used herein, the terms “stably transforming”, “stably transformed”and variations thereof refer to the integration of the exogenous nucleicacid molecules into the genome of the cell such that they aretransferred to progeny cells during cell division without the need forpositively selecting for their presence. Stable transformants, orprogeny thereof, can be selected by any means known in the art such asSouthern blots on chromosomal DNA or in situ hybridization of genomicDNA.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because DNA can be introduced intocells in whole plant tissues or plant organs or explants in tissueculture, for either transient expression or for stable integration ofthe DNA in the plant cell genome. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat.No. 5,104,310, U.S. Pat. No. 5,004,863 or U.S. Pat. No. 5,159,135). Theregion of DNA to be transferred is defined by the border sequences, andthe intervening DNA (T-DNA) is usually inserted into the plant genome.Further, the integration of the T-DNA is a relatively precise processresulting in few rearrangements. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.Preferred Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203(1985).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. An illustrative embodiment of a method for delivering DNA intoZea mays cells by acceleration is a biolistics α-particle deliverysystem, that can be used to propel particles coated with DNA through ascreen, such as a stainless steel or Nytex screen, onto a filter surfacecovered with corn cells cultured in suspension. A particle deliverysystem suitable for use with the present invention is the heliumacceleration PDS-1000/He gun available from Bio-Rad Laboratories.

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain up to 1000 or more foci ofcells transiently expressing a marker gene. The number of cells in afocus that express the exogenous gene product 48 hours post-bombardmentoften range from one to ten and average one to three.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

In another alternative embodiment, plastids can be stably transformed.Methods disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402,5,932,479, and WO 99/05265).

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions that influence the physiological state of therecipient cells and that may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908); soybean (U.S.Pat. Nos. 5,569,834, 5,416,011); Brassica (U.S. Pat. No. 5,463,174);peanut (Cheng et al., 1996); and pea (Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example, CA2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447,PCT/US97/10621, U.S. Pat. Nos. 5,589,617, 6,541,257, and other methodsare set out in Patent specification WO99/14314. Preferably, transgenicwheat or barley plants are produced by Agrobacterium tumefaciensmediated transformation procedures. Vectors carrying the desired nucleicacid construct may be introduced into regenerable wheat cells of tissuecultured plants or explants, or suitable plant systems such asprotoplasts.

The regenerable wheat cells are preferably from the scutellum ofimmature embryos, mature embryos, callus derived from these, or themeristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

A transgenic plant formed using Agrobacterium or other transformationmethods typically contains a single genetic locus on one chromosome.Such transgenic plants can be referred to as being hemizygous for theadded gene(s). More preferred is a transgenic plant that is homozygousfor the added gene(s); i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by self-fertilisinga hemizygous transgenic plant, germinating some of the seed produced andanalyzing the resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants thatcontain two independently segregating exogenous genes or loci can alsobe crossed (mated) to produce offspring that contain both sets of genesor loci. Selfing of appropriate F1 progeny can produce plants that arehomozygous for both exogenous genes or loci. Back-crossing to a parentalplant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Transgenic Non-Human Animals

A “transgenic non-human animal” refers to an animal, other than a human,that contains a gene construct (“transgene”) not found in a wild-typeanimal of the same species or breed. A “transgene” as referred to inthis context has the normal meaning in the art of biotechnology andincludes a genetic sequence which has been produced or altered byrecombinant DNA or RNA technology and which has been introduced into ananimal cell. The transgene may include genetic sequences derived from ananimal cell, which may be of the same or different species or breed asthe cell into which the transgene is introduced. Typically, thetransgene has been introduced into the animal by human manipulation suchas, for example, by transformation but any method can be used as one ofskill in the art recognizes.

Techniques for producing transgenic animals are well known in the art. Auseful general textbook on this subject is Houdebine, Transgenicanimals—Generation and Use (Harwood Academic, 1997). Transformation of apolynucleotide molecule into a cell can be accomplished by any method bywhich a polynucleotide molecule can be inserted into the cell.Transformation techniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and cellfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed polynucleotidemolecules can remain extrachromosomal or can integrate into one or moresites within a chromosome of the transformed (i.e., recombinant) cell insuch a manner that their ability to be expressed is retained.Heterologous DNA can be introduced, for example, into fertilizedmammalian ova. For instance, totipotent or pluripotent stem cells can betransformed by microinjection, calcium phosphate mediated precipitation,liposome fusion, retroviral infection or other means, the transformedcells are then introduced into the embryo, and the embryo then developsinto a transgenic animal. In a highly preferred method, developingembryos are infected with a retrovirus containing the desired DNA, andtransgenic animals produced from the infected embryo. In a mostpreferred method, however, the appropriate DNAs are coinjected into thepronucleus or cytoplasm of embryos, preferably at the single cell stage,and the embryos allowed to develop into mature transgenic animals.

Another method used to produce a transgenic animal involvesmicroinjecting a nucleic acid into pro-nuclear stage eggs by standardmethods. Injected eggs are then cultured before transfer into theoviducts of pseudopregnant recipients.

Transgenic animals may also be produced by nuclear transfer technology.Using this method, fibroblasts from donor animals are stably transfectedwith a plasmid incorporating the coding sequences for a binding domainor binding partner of interest under the control of regulatorysequences. Stable transfectants are then fused to enucleated oocytes,cultured and transferred into female recipients.

Enhancing Exogenous RNA Levels and Stabilized Expression

Silencing Suppressors

Post-transcriptional gene silencing (PTGS) is a nucleotidesequence-specific defense mechanism that can target both cellular andviral mRNAs for degradation PTGS occurs in plants or fungi stably ortransiently transformed with foreign (heterologous) or endogenous DNAand results in the reduced accumulation of RNA molecules with sequencesimilarity to the introduced nucleic acid.

It has widely been considered that co-expression of a silencingsuppressor with a transgene of interest will increase the levels of RNApresent in the cell transcribed from the transgene. Whilst this hasproven true for cells in vitro, significant side-effects have beenobserved in many whole plant co-expression studies. More specifically,as described in Mallory et al. (2002), Chapman et al. (2004), Chen etal. (2004), Dunoyer et al. (2004), Zhang et al. (2006), Lewsey et al.(2007) and Meng et al. (2008) plants expressing silencing suppressors,generally under constitutive promoters, are often phenotypicallyabnormal to the extent that they are not useful for commercialproduction.

As outlined above, the present inventors have found that RNA moleculelevels can be increased, and/or RNA molecule levels stabilized overnumerous generations, by limiting the expression of the silencingsuppressor to a storage organ of a plant or part thereof. As usedherein, a “silencing suppressor” is any polynucleotide or polypeptidethat can be expressed in a plant cell that enhances the level ofexpression product from a different transgene in the plant cell,particularly over repeated generations from the initially transformedplant. In an embodiment, the silencing suppressor is a viral silencingsuppressor or mutant thereof. A large number of viral silencingsuppressors are known in the art and include, but are not limited toP19, V2, P38, Pe-Po and RPV-P0. In an embodiment, the viral silencingsuppressor comprises amino acids having a sequence as provided in anyone of SEQ ID NOs 97 to 101, a biologically active fragment thereof, oran amino acid sequence which is at least 50% identical to any one ormore of SEQ ID NOs 97 to 101 and which has activity as a silencingsuppressor.

As used herein, the terms “stabilising expression”, “stably expressed”,“stabilised expression” and variations thereof refer to level of the RNAmolecule being essentially the same or higher in progeny plants overrepeated generations, for example at least three, at least five or atleast 10 generations, when compared to isogenic plants lacking theexogenous polynucleotide encoding the silencing suppressor. However,this term(s) does not exclude the possibility that over repeatedgenerations there is some loss of levels of the RNA molecule whencompared to a previous generation, for example not less than a 10% lossper generation.

The suppressor can be selected from any source e.g. plant, viral, mammaletc. The suppressor may be, for example:

-   -   flock house virus B2;    -   pothos latent virus P14;    -   pothos latent virus AC2;    -   African cassava mosaic virus AC4;    -   bhendi yellow vein mosaic disease C2;    -   bhendi yellow vein mosaic disease C4;    -   bhendi yellow vein mosaic disease βC1;    -   tomato chlorosis virus p22;    -   tomato chlorosis virus CP;    -   tomato chlorosis virus CPm;    -   tomato golden mosaic virus AL2;    -   tomato leaf curl Java virus βC1    -   tomato yellow leaf curl virus V2;    -   tomato yellow leaf curl virus-China C2    -   tomato yellow leaf curl China virus Y10 isolate βC1;    -   tomato yellow leaf curl Israeli isolate V2;    -   mungbean yellow mosaic virus-Vigna AC2;    -   hibiscus chlorotic ringspot virus CP;    -   turnip crinkle virus P38;    -   turnip crinkle virus CP;    -   cauliflower mosaic virus P6;    -   beet yellows virus p21;    -   citrus tristeza virus p20;    -   citrus tristeza virus p23;    -   citrus tristeza virus CP;    -   cowpea mosaic virus SCP;    -   sweet potato chlorotic stunt virus p22    -   cucumber mosaic virus 2b;    -   tomato aspermy virus HC-Pro    -   beet curly top virus L2;    -   soil borne wheat mosaic virus 19K;    -   barley stripe mosaic virus Gammab;    -   poa scmilatent virus Gammab;    -   peanut clump pecluvirus P15;    -   rice dwarf virus Pns10;    -   curubit aphid borne yellows virus P0;    -   beet western yellows virus P0;    -   potato virus X P25;    -   cucumber vein yellowing virus P1b;    -   plum pox virus HC-Pro;    -   sugarcane mosaic virus HC-Pro    -   potato virus Y strain HC-Pro;    -   tobacco etch virus P1/HC-Pro;    -   turnip mosaic virus P1/HC-Pro;    -   cocksfoot mottle virus P1;    -   cocksfoot mottle virus-Norwegian isolate P1    -   rice yellow mottle virus P1;    -   rice yellow mottle virus-Nigerian isolate P1;    -   rice hoja blanca virus NS3    -   rice stripe virus NS3    -   crucifer infecting tobacco mosaic virus 126K;    -   crucifer infecting tobacco mosaic virus p122;    -   tobacco mosaic virus p122;    -   tobacco mosaic virus 126    -   tobacco mosaic virus 130K;    -   tobacco rattle virus 16K;    -   tomato bushy stunt virus P19;    -   tomato spotted wilt virus NSs;    -   apple chlorotic leaf spot virus P50;    -   grapevine virus A p10;    -   grapevine leafroll associated virus-2 homolog of BYV p21,        as well as variants/mutants thereof. The list above provides the        virus from which the suppressor can be obtained and the protein        (e.g. B2, P14 etc) or coding region designation for the        suppressor from each particular virus.

Multiple copies of a suppressor may be used. Different suppressors maybe used together (e. g., in tandem).

RNA Molecules

Essentially any RNA molecule which is desirable to be expressed in aplant storage organ can be co-expressed with the silencing suppressor.The RNA molecule may influence an agronomic trait, insect resistance,disease resistance, herbicide resistance, sterility, graincharacteristics, and the like. The encoded polypeptides may be involvedin metabolism of oil, starch, carbohydrates, nutrients, etc., or may beresponsible for the synthesis of proteins, peptides, fatty acids,lipids, waxes, oils, starches, sugars, carbohydrates, flavors, odors,toxins, carotenoids. hormones, polymers, flavonoids, storage proteins,phenolic acids, alkaloids, lignins, tannins, celluloses, glycoproteins,glycolipids, etc.

In a particular example, the plants produced increased levels of enzymesfor oil production in plants such as Brassicas, for example oilseed rapeor sunflower, safflower, flax, cotton, soya bean or maize; enzymesinvolved in starch synthesis in plants such as potato, maize, andcereals such as wheat barley or rice; enzymes which synthesize, orproteins which are themselves, natural medicaments, such aspharmaceuticals or veterinary products.

Types of polypeptides that are contemplated for production in a methodof the present invention include pharmaceutical proteins for use inmammals, including man, such as insulin, preproinsulin, proinsulin,glucagon, interferons such as α-interferon and γ-interferon,blood-clotting factors such as Factor VII, VIII, IX, X, XI, and XII,fertility hormones such as luteinising hormone, follicle stimulatinghormone growth factors such as epidermal growth factor, platelet-derivedgrowth factor, granulocyte colony stimulating factor, prolactin,oxytocin, thyroid stimulating hormone, adrenocorticotropic hormone,calcitonin, parathyroid hormone, somatostatin, erythropoietin (EPO),enzymes such as β-glucocerebrosidase, haemoglobin, serum albumin,collagen, growth hormone, human serum albumin, human-secreted alkalinephosphatase, aprotinin, al-antitrypsin, IgG1 (phosphonate ester), IgM(neuropeptide hapten), SIgA/G (Streptococcus mutans adhesin),scFv-bryodin 1 immunotoxin (CD 40), IgG (HSV), LSC (HSV) and the like.

Furthermore, the method of the invention can be used for the productionof specific antibodies, including antibody-related molecules or activefragments thereof which bind, for example, bone morphogenetic proteinreceptor-type IB; E16; STEAP1; MPF; Napi3b; Sema 5b; PSCA; Endothelintype B receptor; MSG783; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2;HER2; NCA; MDP; IL20Rα; Brevican; EphB2R; ASLG659; PSCA; GEDA; Bcell-activating factor receptor; CD22; CD79a; CXCR5; HLA-DOB; P2X5;CD72; LY64; FcRH1; IRTΔ2; TENB2; CD20; VEGF including VEGF_A, B, C or D;p53; EGFR; progesterone receptor; cathepsin D; Bcl-2; E cadherin; CEA;Lewis X; Ki67; PCNA; CD3; CD4; CD5; CD7; CD11c; CD11d; c-Myc; tau;PrPSC; or Aβ

In addition, the method of the invention can be used for the productionof an antigen, which may or may not be delivered by consumption of thestorage organ, examples of which include Hepatitis B virus envelopeprotein, rabies virus glycoprotein, Escherichia coli heat-labileentertoxin, Norwalk virus capsid protein, diabetes autoantigen, choleratoxin B subunit, cholera toxin B and Δ2 subunits, rotavirus entertoxinand enterotoxigenic E. coli fimbrial antigen fusions, porcinetransmissible gastroenteritis virus glycoprotein S, human rhinovirus 15(HRV-14) and human immunodeficiency virus type (HIV-1) epitopes, MinkEnteritis Virus epitopes, foot and mouth disease virus VP1 structuralprotein, human cytomegalovirus glycoprotein B, dental caries (S. mutans)antigens, and respiratory syncytial virus antigens.

Levels of LC-PUFA Produced

The levels of the LC-PUFA or combination of LC-PUFAs that are producedin the recombinant cell are of importance. The levels may be expressedas a composition (in percent) of the total fatty acid that is aparticular LC-PUFA or group of related LC-PUFA, for example the ω3LC-PUFA or the ω6 LC-PUFA, or the VLC-PUFA, or other which may bedetermined by methods known in the art. The level may also be expressedas a LC-PUFA content, such as for example the percentage of LC-PUFA inthe dry weight of material comprising the recombinant cells, for examplethe percentage of the dry weight of seed that is LC-PUFA. It will beappreciated that the LC-PUFA that is produced in an oilseed may beconsiderably higher in terms of LC-PUFA content than in a vegetable or agrain that is not grown for oil production, yet both may have similarLC-PUFA compositions, and both may be used as sources of LC-PUFA forhuman or animal consumption.

The levels of LC-PUFA may be determined by any of the methods known inthe art. In a preferred method, total lipid is extracted from the cells,tissues or organisms and the fatty acid converted to methyl estersbefore analysis by gas chromatography (GC). Such techniques aredescribed in Example 1. The peak position in the chromatogram may beused to identify each particular fatty acid, and the area under eachpeak integrated to determine the amount. As used herein, unless statedto the contrary, the percentage of particular fatty acid in a sample isdetermined as the area under the peak for that fatty acid as apercentage of the total area for fatty acids in the chromatogram. Thiscorresponds essentially to a weight percentage (w/w). The identity offatty acids may be confirmed by GC-MS. Total lipid may be separated bytechniques known in the art to purify fractions such as the TAGfraction. For example, thin-layer chromatography (TLC) may be performedat an analytical scale to separate TAG from other lipid fractions suchas DAG, acyl-CoAs or phospholipid in order to determine the fatty acidcomposition specifically of TAG.

In one embodiment, the sum total of ARA, EPA, DPA and DHA in the fattyacids in the cell comprises at least 15%, more preferably at least 20%or at least 25% of the total fatty acids in the cell. In a morepreferred embodiment, the sum total of those fatty acids is at least29%, at least 30% or at least 31% of the total fatty acids in the cell.In a further embodiment, the total fatty acid in the cell has less than1% C20:1. In another embodiment, the amount of DHA in the fatty acids inthe cell is at least 3%, more preferably at least 4%, more preferably atleast 5% or at least 7%, or most preferably at least 10%, of the totalfatty acids in the cell. In preferred embodiments, the extractable TAGin the cell comprises the fatty acids at the levels referred to in thisparagraph. Each possible combination of these features is alsoencompassed. For example, the sum total of ARA, EPA, DPA and DHA in thefatty acids in the cell may comprises at least 15%, at least 20%, atleast 25%, at least 29%, at least 30% or at least 31% of the total fattyacids in the cell, of which at least 3%, at least 4%, at least 5%, atleast 7% or at least 10% of the total fatty acids in the cell is DHA,while the level of C20:1 may be less than 1%.

In each of these embodiments, the recombinant cell may be a cell of anorganism that is suitable for fermentation such as, for example, aunicellular microorganism which may be a prokaryote or a eukaryote suchas yeast, or a plant cell. In a preferred embodiment, the cell is a cellof an angiosperm (higher plant). In a further preferred embodiment, thecell is a cell in a seed such as, for example, an oilseed or a grain orcereal.

The level of production of LC-PUFA in the recombinant cell may also beexpressed as a conversion ratio, i.e., the amount of the LC-PUFA formedas a percentage of one or more substrate PUFA or LC-PUFA. With regard toEPA, for example, this may be expressed as the ratio of the level of EPA(as a percentage in the total fatty acid) to the level of a substratefatty acid (ALA, SDA, ETA or ETrA).

In one embodiment, the efficiency of conversion of ALA to EPA is atleast 80%, or more preferably at 90%. In another embodiment, theefficiency of conversion of ALA to EPA, DPA or DHA (calculated as thesum of the percentages for EPA, DPA and DHA/the sum of the percentagesfor ALA and all Δ6-desaturated fatty acid products from ALA) is at least17.3%, or at least 23%. In another embodiment, the efficiency ofconversion of ALA to DPA or DHA (calculated as the sum of thepercentages for DPA and DHA/the sum of the percentages for ALA and allΔ6-desaturated fatty acid products from ALA) is at least 15.4%, or atleast 21%. In another embodiment, the efficiency of conversion of ALA toDHA (calculated as the percentage for DHA/the sum of the percentages forALA and all Δ6-desaturated fatty acid products from ALA) is at least9.5%, or at least 10.8%. In another embodiment, the efficiency ofconversion of EPA to DHA (calculated as the percentage for DHA/the sumof the percentages for EPA and all Δ5-elongated fatty acid products fromEPA) is at least 45%, or at least 50%. In another embodiment, theefficiency of conversion of SDA to produce ETA (calculated as the sum ofthe percentages for ETA and Δ5-desaturated fatty acid products fromETA/the sum of the percentages for SDA and all Δ6-elongated fatty acidproducts from SDA) is at least 50%, more preferably at least 60%. Inanother embodiment, the efficiency of conversion of ALA to ETrA is atleast 6%, more preferably at least 9%. In another embodiment, theconversion efficiency of EPA to DPA (calculated as the sum of thepercentages for DPA and DHA/the sum of the percentages for EPA, DPA andDHA) through a Δ5 elongase step is at least 60%, more preferably atleast 65%, more preferably at least 70% or most preferably at least 75%.

The content of the LC-PUFA in the recombinant cell may be maximized ifthe parental cell used for introduction of the genes is chosen such thatthe level of fatty acid substrate that is produced or providedexogenously is optimal. The level of LC-PUFA may also be maximized bygrowing or incubating the cells under optimal conditions, for example ata slightly lower temperature than the standard temperature for thatcell, which is thought to favour accumulation of polyunsaturated fattyacid. In particular however, evidence to date suggests that somedesaturases expressed heterologously in yeast or plants have relativelylow activity in combination with some elongases. This may be alleviatedby providing a desaturase with the capacity of to use an acyl-CoA formof the fatty acid as a substrate in LC-PUFA synthesis, and this isthought to be advantageous in recombinant cells other than yeast such asplant cells.

Production of Oils

Techniques that are routinely practiced in the art can be used toextract, process, and analyze the oils produced by cells, plants, seeds,etc of the instant invention. Typically, plant seeds are cooked,pressed, and extracted to produce crude oil, which is then degummed,refined, bleached, and deodorized. Generally, techniques for crushingseed are known in the art. For example, oilseeds can be tempered byspraying them with water to raise the moisture content to, e.g., 8.5%,and flaked using a smooth roller with a gap setting of 0.23 to 0.27 mm.Depending on the type of seed, water may not be added prior to crushing.Application of heat deactivates enzymes, facilitates further cellrupturing, coalesces the oil droplets, and agglomerates proteinparticles, all of which facilitate the extraction process.

The majority of the seed oil is released by passage through a screwpress. Cakes expelled from the screw press are then solvent extracted,e.g., with hexane, using a heat traced column. Alternatively, crude oilproduced by the pressing operation can be passed through a settling tankwith a slotted wire drainage top to remove the solids that are expressedwith the oil during the pressing operation. The clarified oil can bepassed through a plate and frame filter to remove any remaining finesolid particles. If desired, the oil recovered from the extractionprocess can be combined with the clarified oil to produce a blendedcrude oil.

Once the solvent is stripped from the crude oil, the pressed andextracted portions are combined and subjected to normal oil processingprocedures (i.e., degumming, caustic refining, bleaching, anddeodorization). Degumming can be performed by addition of concentratedphosphoric acid to the crude oil to convert non-hydratable phosphatidesto a hydratable form, and to chelate minor metals that are present. Gumis separated from the oil by centrifugation. The oil can be refined byaddition of a sufficient amount of a sodium hydroxide solution totitrate all of the fatty acids and removing the soaps thus formed.

Deodorization can be performed by heating the oil to 260° C. undervacuum, and slowly introducing steam into the oil at a rate of about 0.1ml/minute/100 ml of oil. After about 30 minutes of sparging, the oil isallowed to cool under vacuum. The oil is typically transferred to aglass container and flushed with argon before being stored underrefrigeration. If the amount of oil is limited, the oil can be placedunder vacuum, e.g., in a Parr reactor and heated to 260° C. for the samelength of time that it would have been deodorized. This treatmentimproves the color of the oil and removes a majority of the volatilesubstances.

Feedstuffs

The present invention includes compositions which can be used asfeedstuffs. For purposes of the present invention, “feedstuffs” includeany food or preparation for human or animal consumption (including forenteral and/or parenteral consumption) which when taken into the body(a) serve to nourish or build up tissues or supply energy; and/or (b)maintain, restore or support adequate nutritional status or metabolicfunction. Feedstuffs of the invention include nutritional compositionsfor babies and/or young children.

Feedstuffs of the invention comprise, for example, a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product ofthe method of the invention, the product of the fermentation process ofthe invention, or a composition along with a suitable carrier(s). Theterm “carrier” is used in its broadest sense to encompass any componentwhich may or may not have nutritional value. As the skilled addresseewill appreciate, the carrier must be suitable for use (or used in asufficiently low concentration) in a feedstuff such that it does nothave deleterious effect on an organism which consumes the feedstuff.

The feedstuff of the present invention comprises an oil, fatty acidester, or fatty acid produced directly or indirectly by use of themethods, cells or plants disclosed herein. The composition may either bein a solid or liquid form. Additionally, the composition may includeedible macronutrients, vitamins, and/or minerals in amounts desired fora particular use. The amounts of these ingredients will vary dependingon whether the composition is intended for use with normal individualsor for use with individuals having specialized needs, such asindividuals suffering from metabolic disorders and the like.

Examples of suitable carriers with nutritional value include, but arenot limited to, macronutrients such as edible fats, carbohydrates andproteins. Examples of such edible fats include, but are not limited to,coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and diglycerides. Examples of such carbohydrates include (but arenot limited to): glucose, edible lactose, and hydrolyzed search.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include (but are not limitedto) soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thefeedstuff compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the presentinvention can be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by de novo synthesis.

A feedstuff composition of the present invention may also be added tofood even when supplementation of the diet is not required. For example,the composition may be added to food of any type, including (but notlimited to): margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

The genus Saccharomyces spp is used in both brewing of beer and winemaking and also as an agent in baking, particularly bread. Yeast is amajor constituent of vegetable extracts. Yeast is also used as anadditive in animal feed. It will be apparent that genetically engineeredyeast strains can be provided which are adapted to synthesise LC-PUFA asdescribed herein. These yeast strains can then be used in food stuffsand in wine and beer making to provide products which have enhancedfatty acid content.

Additionally, fatty acids produced in accordance with the presentinvention or host cells transformed to contain and express the subjectgenes may also be used as animal food supplements to alter an animal'stissue or milk fatty acid composition to one more desirable for human oranimal consumption. Examples of such animals include sheep, cattle,horses and the like.

Furthermore, feedstuffs of the invention can be used in aquaculture toincrease the levels of fatty acids in fish for human or animalconsumption.

Preferred feedstuffs of the invention are the plants, seed and otherplant parts such as leaves and stems which may be used directly as foodor feed for humans or other animals. For example, animals may grazedirectly on such plants grown in the field or be fed more measuredamounts in controlled feeding. The invention includes the use of suchplants and plant parts as feed for increasing the LC-PUFA levels inhumans and other animals.

Compositions

The present invention also encompasses compositions, particularlypharmaceutical compositions, comprising one or more of the fatty acidsand/or resulting oils produced using the methods of the invention.

A pharmaceutical composition may comprise one or more of the fatty acidsand/or oils, in combination with a standard, well-known, non-toxicpharmaceutically-acceptable carrier, adjuvant or vehicle such asphosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquidor powder, injectible, or topical ointment or cream. Proper fluidity canbe maintained, for example, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. It mayalso be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Besides such inert diluents, thecomposition can also include adjuvants, such as wetting agents,emulsifying and suspending agents, sweetening agents, flavoring agentsand perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, fatty acids produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant fatty acid(s).

For intravenous administration, the fatty acids produced in accordancewith the present invention or derivatives thereof may be incorporatedinto commercial formulations.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g,taken from one to five times per day (up to 100 g daily) and ispreferably in the range of from about 10 mg to about 1, 2, 5, or 10 gdaily (taken in one or multiple doses). As known in the art, a minimumof about 300 mg/day of fatty acid, especially LC-PUFA, is desirable.However, it will be appreciated that any amount of fatty acid will bebeneficial to the subject.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include, for example, enteral (e.g., oral andrectal) and parenteral. For example, a liquid preparation may beadministered orally or rectally. Additionally, a homogenous mixture canbe completely dispersed in water, admixed under sterile conditions withphysiologically acceptable diluents, preservatives, buffers orpropellants to form a spray or inhalant.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, overallhealth of the patient, past history of the patient, immune status of thepatient, etc.

Additionally, the compositions of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or a fatty acid producedaccording to the subject invention may be used as the sole “active”ingredient in a cosmetic composition.

EXAMPLES Example 1 Materials and Methods

Culturing Microalgae

Micromonas CS-0170 and Pyramimonas CS-0140 isolates from the CSIROCollection of Living Microalgae (http://www.marine.csiro.au/microalgae)were cultivated under standard culture conditions. A stock culture fromthe Collection was sub-cultured and scaled-up in a dilution of 1 in 10over consecutive transfers in 1 L Erlenmeyer flasks and then into 10 Lpolycarbonate carboys. The culture medium was f/2, a modification ofGuillard and Ryther's (1962) f medium containing half-strengthnutrients, with a growth temperature of 20±1° C. Other culturingconditions included a light intensity of 100 μmol. photons PAR.m-2.s-1,12:12 hour light:dark photoperiod, and bubbling with 1% CO₂ in air at arate of 200 mL·L⁻¹·min⁻¹.

Isolation of Microalgal Genomic DNA

Genomic DNA from Micromonas CS-0170 and Pyramimonas CS-0140 was isolatedusing the DNeasy Plant Mini Kit system as described in the accompanyinginstruction manual (QIAGEN, catalogue #69106).

Isolation of Microalgal Total RNA

Total RNA was isolated from Micromonas CS-0170 and Pyramimonas CS-0140cells using the following method. 2 g (wet weight) of cells werepowdered using a mortar and pestle in liquid nitrogen and sprinkledslowly into a beaker containing 22 mL of extraction buffer that wasbeing stirred constantly. To this, 5% insoluble polyvinylpyrrolidone, 90mM 2-mercaptoethanol, and 10 mM dithiothreitol were added and themixture stirred for a further 10 minutes prior to being transferred to aCorex™ tube. 18.4 mL of 3 M ammonium acetate was added and mixed well.The sample was then centrifuged at 6000×g for 20 minutes at 4° C. Thesupernatant was transferred to a new tube and nucleic acid precipitatedby the addition of 0.1 volume of 3 M NaAc (pH 5.2) and 0.5 volume ofcold isopropanol. After a 1 hour incubation at −20° C., the sample wascentrifuged at 6000×g for 30 minutes in a swing rotor. The pellet wasresuspended in 1 mL of water extracted with phenol/chloroform. Theaqueous layer was transferred to a new tube and nucleic acids wereprecipitated once again by the addition of 0.1 volume 3 M NaAc (pH 5.2)and 2.5 volumes of ice cold ethanol. The pellet was resuspended in waterand the concentration of nucleic acid determined by spectrophotometer.

Vectors and Strains

Plasmid pYES2 and yeast strain INVSC1 were obtained from Invitrogen,plasmid vector pGEMT-Easy from Promega, plasmid vector pBluescript IIKS— from Stratagene. Agrobacterium tumefaciens strain AGL1 was referredto by Lazo et al. (1991) and the pORE binary vector series by Coutu etal. (2007).

PCR Conditions

To amplify DNA fragments by polymerase chain reaction (PCR), standardconditions were used unless specified otherwise. Optimisation ofconditions was carried out by varying the number of amplificationcycles, the temperature for annealing of the primers, Mg²⁺ concentrationand other parameters as is typically done in the art. Buffers were asspecified by the suppliers of the polymerases. Typically, reactionconditions were as follows. After an initial denaturation at 94° C. for2-3 min, reaction mixtures were treated for 20-40 cycles ofdenaturation/annealing/extension with denaturation at 94° C. for 30-60sec, primer annealing at 40-60° C. for 30 sec, and polymerase extensionfor 30-60 sec at 70-72° C., followed by a further extension step of 3min at 70-72° C.

Reverse transcription-PCR (RT-PCR) amplification was typically carriedout using the Superscript III One-Step RT-PCR system (Invitrogen) in avolume of 25 μL using 10 pmol of the forward primer and 30 pmol of thereverse primer, MgSO₄ to a final concentration of 2.5 mM, 400 ng oftotal RNA with buffer and nucleotide components according to themanufacturer's instructions. Typical temperature regimes were: 1 cycleof 45° C. for 30 minutes for the reverse transcription to occur; then 1cycle of 94° C. for 2 minutes followed by 40 cycles of 94° C. for 30seconds, 52° C. for 30 seconds, 70° C. for 1 minute; then 1 cycle of 72°C. for 2 minutes before cooling the reaction mixtures to 5° C.

5′ and 3′-RACE

To obtain full length cDNAs corresponding to partial length genefragments, the 5′ and/or 3′ ends of cDNAs were obtained by 5′- and3′-RACE (Rapid Amplification of cDNA Ends) methods. The 3′ end of a cDNAwas isolated using a gene specific forward primer as specified in theExamples and an oligo-dT reverse primer5′-ATTTAGGTGACACTATAGTTTTTTTTTTTTTTTTTTV-3′ (SEQ ID NO:41), where Vrepresents either A, G or C, which was in common to all of the 3′-RACEreactions. An RT-PCR amplification was carried out using the SuperscriptIII One-Step RT-PCR system (Invitrogen) in a volume of 25 μL using 10pmol of the forward primer and 30 pmol of the reverse primer, MgSO₄ to afinal concentration of 2.5 mM, 400 ng of total RNA as template for cDNAsynthesis, and buffer and nucleotide components as specified by thesupplier. The cycling conditions were typically: 1 cycle of 45° C. for30 minutes for reverse transcription; then 1 cycle of 94° C. for 2minutes; followed by 40 cycles of 94° C. for 30 seconds, 52° C. for 30seconds, 70° C. for 1 minute and 1 cycle of 72° C. for 2 minutes beforecooling to 5° C. The amplicons generated in the reaction were ligatedinto pGEM-T Easy, cloned into E. coli and sequenced by standard methods.

Unless specified otherwise, the 5′ end of cDNAs were isolated using amodified terminal-transferase method with 2 μg of total RNA as templatefor cDNA synthesis. 10 pmol of a gene specific reverse primer was addedto the total RNA and 10.8 μL water before the mixture was heated at 65°C. for 5 minutes and chilled on ice for 2 minutes. The followingcomponents were then added: 4 μL of Superscript III first-strand cDNAbuffer (Invitrogen), 1 μL of 0.1 M dithiothreitol, 1 μL RNAseOUT(Invitrogen) and 1 μL of Superscript III reverse transcriptase. Themixture was then incubated at 55° C. for 60 minutes and the reactionterminated by a further incubation at 70° C. for 15 minutes. After beingcooled briefly on ice the reaction was then treated with 2 units ofRNAseH at 37° C. for 20 minutes. The cDNA was then purified using theQIAQUICK PCR Purification Kit (QIAGEN, catalogue #28106). 25 μL of theeluate was then A-tailed using 10 units of TdT (NEB), 5 μL of NEB Buffer#4, 5 μL of 2.5 mM CoCl₂, 0.5 μL of 10 mM dATP in a total of 50 μL. Thereaction was performed at 37° C. for 30 minutes followed by inactivationof the enzyme at 70° C. for 10 minutes. A PCR reaction was thenperformed using 2.5 units of Taq DNA polymerase (NEB) in the reactionmixture including 5 μL of the A-tailed cDNA, 10 pmol of the genespecific reverse primer, 30 pmol of a modified oligo-dT primer5′-ATTTAGGTGACACTATAGTTTTTTTTTTTTTTTTTTV-3′ (SEQ ID NO:41), where Vrepresents either A, G or C, and buffer and nucleotide components asspecified in the accompanying manual. The cycling conditions weretypically: 1 cycle of 94° C. for 2 minutes; 5 cycles of 94° C. for 20seconds, 54° C. for 1 minute, 72° C. for 1 minute; 30 cycles of 94° C.for 20 seconds, 60° C. for 30 seconds, 72° C. for 1 minute; 1 cycle of72° C. for 5 minutes; 4° C. hold. If no clear product band was visiblein the expected size range after gel electrophoresis, the region of thegel was excised and DNA products purified from the gel. A sample of 1 μLof a 1:20 dilution of the eluate was used as template in a second roundof PCR. The amplicons generated in the reaction were ligated into pGEM-TEasy and sequenced.

Yeast Culturing and Feeding with Precursor Fatty Acids

Plasmids were introduced into yeast by heat shock and transformants wereselected on yeast minimal medium (YMM) plates containing 2% raffinose asthe sole carbon source. Clonal inoculum cultures were established inliquid YMM with 2% raffinose as the sole carbon source. Experimentalcultures in were inoculated from these, in YMM+1% NP-40, to an initialOD600 of ˜0.3. Cultures were grown at 30° C. with shaking (˜60 rpm)until OD600 was approximately 1.0. At this point galactose was added toa final concentration of 2% and precursor fatty acids were added to afinal concentration of 0.5 mM. Cultures were incubated at 20° C. withshaking for a further 48 hours prior to harvesting by centrifugation.Cell pellets were washed with 1% NP-40, 0.5% NP-40 and water to removeany unincorporated fatty acids from the surface of the cells.

Expression of Genes in Plant Cells in a Transient Expression System

Genes were expressed in plant cells in a transient expression systemessentially as described by Voinnet et al. (2003). Plasmids containingthe coding region to be expressed from a strong constitutive promotersuch as the 35S promoter were introduced into Agrobacterium tumefaciensstrain AGL1. A chimeric gene 35S:p19 for expression of the p19 viralsilencing suppressor was separately introduced into AGL1. Therecombinant cells were grown at 28° C. in LB broth supplemented with 50mg/mL kanamycin and 50 mg/mL rifampicin to stationary phase. Thebacteria were then pelleted by centrifugation at 5000 g for 15 min atroom temperature before being resuspended to OD600=1.0 in aninfiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl₂ and 100 uMacetosyringone. The cells were then incubated at 28° C. with shaking for3 hours before equal volumes of Agrobacterium cultures containing35S:p19 and the test chimeric gene(s) of interest were mixed prior toinfiltration into leaf tissue. The plants were typically grown for afurther five days after infiltration before leaf discs were taken for GCanalysis of the fatty acids.

Where leaf tissue was supplied with exogenous fatty acids, the fattyacids were prepared by heating the appropriate fatty acid in 2M ammoniumhydroxide solution for 20 minutes at 60° C. after which the solution wasevaporated, also at 60° C. The resulting salt was then resuspended in0.1M phosphate buffer (pH 7.2) to a final concentration of 0.5 μg/mL.The fatty acid salt was injected into the leaf four days afterAgrobacterium infiltration and leaf discs taken at various time pointsafter feeding, for example from 2-48 hours after addition of theexogenous fatty acid, for analysis of the fatty acid composition.Controls were included where the exogenous fatty acid was omitted, orwhere the Agrobacterium strain used for the infiltration did not containthe gene of interest.

Gas Chromatography (GC) Analysis of Fatty Acids

Fatty Acid Preparation

Where a sample contained a large amount of water, including allNicotiana benthamiana leaf samples and other non-seed tissues, the totallipids were extracted using the method described by Bligh and Dyer(1959) prior to methylation. Fatty acid methyl esters (FAME) were formedby transesterification of the centrifuged yeast pellet, Arabidopsisseeds, total lipids from Nicotiana benthamiana or other total lipidsamples by heating with MeOH—CHCl₃—HCl (10:1:1, v/v/v) at 90-100° C. for2 hours in a glass test tube fitted with a Teflon-lined screw-cap. FAMEwere extracted into hexane-dichloromethane (4:1, v/v) and analysed by GCand GC-MS.

Capillary Gas-liquid Chromatography (GC)

FAME were analysed by gas chromatography (GC) using an AgilentTechnologies 6890N GC (Palo Alto, Calif., USA) equipped with anEquity™-1 fused silica capillary column (15 m×0.1 mm i.d., 0.1 μm filmthickness), an FID, a split/splitless injector and an AgilentTechnologies 7683 Series auto sampler and injector. Helium was used asthe carrier gas. Samples were injected in splitless mode at an oventemperature of 120° C. After injection, the oven temperature was raisedto 270° C. at 10° C.min⁻¹ and finally to 310° C. at 5° C.min⁻¹. Peakswere quantified with Agilent Technologies ChemStation software (RevB.03.01 (317), \ Palo Alto, Calif., USA).

Gas Chromatography-mass Spectrometry (GC-MS)

GC-MS was carried out on a Finnigan GCQ Plus GC-MS ion-trap fitted withon-column injection set at 4° C. Samples were injected using an AS2000auto sampler onto a retention gap attached to an HP-5 Ultra 2bonded-phase column (50 m×0.32 mm i.d.×0.17 μm film thickness). Theinitial temperature of 45° C. was held for 1 minute, followed bytemperature programming at 30° C.min⁻¹ to 140° C. then at 3° C.min⁻¹ to310° C. where it was held for 12 minutes. Helium was used as the carriergas. Mass spectrometer operating conditions were: electron impact energy70 eV; emission current 250 μamp, transfer line 310° C.; sourcetemperature 240° C.; scan rate 0.8 scans.s⁻¹ and mass range 40-650Dalton. Mass spectra were acquired and processed with Xcalibur™software.

Yeast Culturing and Feeding with Precursor Fatty Acids

Plasmids were introduced into yeast by heat shock and transformants wereselected on yeast minimal medium (YMM) plates containing 2% raffinose asthe sole carbon source. Clonal inoculum cultures were established inliquid YMM with 2% raffinose as the sole carbon source. Experimentalcultures in were inoculated from these, in YMM+1% NP-40, to an initialOD₆₀₀ of ˜0.3. Cultures were grown at 30° C. with shaking (˜60 rpm)until OD₆₀₀ was approximately 1.0. At this point galactose was added toa final concentration of 2% and precursor fatty acids were added to afinal concentration of 0.5 mM. Cultures were incubated at 20° C. withshaking for a further 48 hours prior to harvesting by centrifugation.Cell pellets were washed with 1% NP-40, 0.5% NP-40 and water to removeany unincorporated fatty acids from the surface of the cells.

Example 2 Isolation and Characterisation of cDNAs Encoding Δ6-elongasefrom Microalgae

Isolation of a Micromonas CS-0170 Δ6-elongase Gene Fragment

The Micromonas CS-0170 strain in the CSIRO Living Collection ofMicroalgae (WO2005/103253) was identified as a microalgal strain thathad a high native level of Δ5- and Δ6-elongation (Table 4).

TABLE 4 Conversion of fatty acids in the CSIRO Collection of LivingMicroalgac strains Micromonas CS-0170 and Pyramimonas CS-0140. TypePRASINOPHYCEAE PRASINOPHYCEAE Species Micromonas pusilla Pyramimonascordata Strain CS0170 CS0140 Phase logarithmic logarithmic 16:1 (n-7)0.7 0.8 18:1 (n-9) 0.3 0.2 18:1 (n-7) 5.5 14.8 16:2 (n-7) 0.2 0.0 18:2(n-6) 0.1 0.7 18:3 (n-6) 0.0 0.0 20:4 n-6) 0.0 0.0 16:3 (n-3) 0.0 0.016:4 (n-3) 20.4 14.3 18:3 (n-3) 1.4 4.6 18:4 (n-3) 20.7 25.6 18:5 (n-3)16.7 3.4 20:3 (n-3) 0.1 1.2 20:4 (n-3) 0.0 0.0 20:5 (n-3) 0.3 0.4 22:5(n-3) 0.3 4.1 22:6 (n-3) 8.5 4.5

In an attempt to identify conserved sequences, elongase amino acidsequences from GenBank accession numbers AAV67800, ABC18314, CAD58540,CAL55414, AAV67797, XP_001416454, AAW70157, AAV67799, ABC18313, AAY15135were aligned using the ClustalW algorithm. Amongst numerous regions ofhomology of various degrees of identity, the consensus amino acidsequence blocks KXXXXXDT (SEQ ID NO:31) and MYXYY (SEQ ID NO:32) werechosen (where each X is, independently, any amino acid), correspondingto amino acid positions 144-151 and 204-208, respectively, of AAY15135.The degenerate primers 5′-AAGWWCIKSGARYISYTCGACAC-3′ (SEQ ID NO:42) and5′-AIIMIRTARTASGTGTACAT-3′ (SEQ ID NO:43) where I=inosine, W=A or T, R=Aor G, Y=C or T, K=G or T, M=A or C, S=C or G, were synthesised based onthe sequences of these two blocks. An RT-PCR amplification was carriedout using the Superscript III One-Step RT-PCR system (Invitrogen) in avolume of 50 μL using 20 pmol of each primer, MgSO₄ to a finalconcentration of 2.5 mM, 200 ng of Micromonas CS-0170 total RNA withbuffer and nucleotide components as specified. The cycling conditionswere: initial 48° C. for 30 minutes for reverse transcription, then 1cycle of 94° C. for 2 minutes, followed by 5 cycles of 94° C. for 30seconds, 40° C. for 30 seconds, 70° C. for 30 seconds; then 40 cycles of94° C. for 30 seconds, 45° C. for 30 seconds, 70° C. for 30 seconds andthen 72° C. for 2 minutes. A 209 bp amplicon was generated, ligated intopGEM-T Easy and sequenced.

Isolation of a Full Length cDNA Encoding Micromonas CS-0170 Δ6-elongase

Primers were designed to extend the 209 bp fragment by 5′- and 3′-RACE.The 3′ end of the gene was isolated as described in Example 1 using thegene specific forward primer 5′-GAACAACGACTGCATCGACGC-3′ (SEQ ID NO:44)and 200 ng of Micromonas CS-0170 total RNA. A 454 bp amplicon wasgenerated, ligated into pGEM-T Easy and sequenced. The 5′ end of thegene was isolated using the GeneRacer Kit (Invitrogen, catalogue#L1500-01) with a reverse-transcription incubation of 55° C. for 1 hourto generate 5′-adapted cDNA as described in the accompanying manual. TheGeneRacer 5′ Primer 5′-CGACTGGAGCACGAGGACACTGA-3′ (SEQ ID NO:45) and thegene specific reverse primer 5′-TTGCGCAGCACCATAAAGACGGT-3′ (SEQ IDNO:46) were used in a PCR amplification using PFU Ultra II Fusion DNApolymerase in a volume of 50 μL using 10 pmol of each primer, 1 μl ofthe GeneRacer cDNA template with buffer and nucleotide components asspecified by the manufacturer (Stratagene, catalogue #600670). Thecycling conditions were: 1 cycle of 94° C. for 2 minutes; 35 cycles of94° C. for 20 seconds, 55° C. for 30 seconds, 72° C. for 30 seconds;then 72° C. for 2 minutes before cooling to 4° C. This product was thendiluted 1:10 and 1 μl used as template in a second round of PCR usingthe GeneRacer 5′ Nested Primer 5′-GGACACTGACATGGACTGAAGGAGTA-3′ (SEQ IDNO:47) and the gene specific reverse primer5′-TTGCGCAGCACCATAAAGACGGT-3′ (SEQ ID NO:46) using the same PCRconditions as used in the first round of amplification. A 522 bpamplicon was generated, ligated into pGEM-T Easy and sequenced.

The nucleotide sequences of the three amplicons were assembled into onesequence which was predicted to be the full-length sequence. The fulllength coding region with a short region of 5′ UTR was then amplifiedfrom genomic DNA from Micromonas strain CS-0170 using forward primer5′-CAGGCGACG CGCGCCAGAGTCC-3′ (SEQ ID NO:48), reverse primer5′-TTATTAGTTACTTGGCCTTTACCTTC-3′ (SEQ ID NO:49) and PFU Ultra II FusionDNA polymerase (Stratagene). An 860 bp amplicon was generated, ligatedinto pGEM-T Easy and sequenced. The sequence of the open reading frameof the gene is presented in having the sequence of SEQ ID NO:1.

The full-length amino acid sequence encoded by the gene is presented asSEQ ID NO:2. BLAST analysis of the protein sequence revealed that theisolated cDNA encoded either a Δ5- or Δ6-elongase. These two types ofelongases are similar at the amino acid level and it was uncertain fromamino acid sequence alone which activity was encoded. When used as aquery sequence to the Genbank protein sequence database using BLASTP,the maximum degree of identity between the Micromonas CS-0170 elongaseand other elongases was 65% with Accession No. CAL55414 which is thesequence for Ostreococcus tauri polyunsaturated fatty acid elongase 2.The conserved GNS1/SUR4 family domain (NCBI conserved domain pfam01151)is represented in this sequence at amino acids 49 to 274, whichtypically indicates that the protein is involved in long chain fattyacid elongation systems.

A sequence relationship tree based on multiple alignment of sequencessimilar to the Micromonas CS-0170 elongase, including those used todesign the original degenerate primers, is provided in FIG. 3.

Functional Characterisation of the Micromonas CS-0170 Δ6-elongase inYeast

The entire protein coding region of this clone, contained within aSalI/SphI fragment in pGEM-T Easy was inserted into pYES2 at theXhoI/SphI sites, generating vector pYES2+MicElo1 for introduction andfunctional characterisation in yeast. Cells of yeast strain INVSC1 weretransformed with pYES2+MicElo1 and transformants were selected on mediumwithout uracil. The yeast cells containing pYES2+MicElo1 were grown inculture and the GAL promoter induced by galactose for expression of theMicElo1 gene. After the addition of ALA, SDA or EPA (0.5 mM) to theculture medium and 48 hours of further culturing at 30° C. the fattyacids in total cellular lipids were analysed. When ALA was added to themedium the presence of ETrA in the cellular lipid of the yeasttransformants was detected at 0.2% of total fatty acids, representing alow but measurable 0.4% conversion efficiency. Similarly, when SDA wasadded to the medium, the presence of ETA in the cellular lipid of theyeast transformants was detected at 0.2%, representing 0.4% conversionefficiency, indicating a low level of Δ6-elongase activity. However,when EPA was added to the medium, the presence of DPA in the cellularlipid of the yeast transformants was not detected, indicating a lack ofΔ5-elongase activity in the yeast cells (Table 5).

TABLE 5 Conversion of fatty acids in yeast cells transformed withgenetic constructs expressing elongases isolated from Micromonas CS-0170and Pyramimonas CS-0140. Fatty acid precursor/ Fatty acid formed/Conversion Clone % of total FA % of total FA ratio pYES2 + ALA,18:3ω3/52.2% ETrA, 20:3ω3/0.2% 0.4% Mic-Elo1 pYES2 + SDA, 18:4ω3/54.3%ETA, 20:4ω3/0.2% 0.4% Mic-Elo1 pYES2 + EPA, 20:5ω3/2.0% DPA, 22:5ω3/0%  0% Mic-Elo1 pYES2 + ALA, 18:3ω3/51.4% ETrA, 20:3ω3/5.3% 9.3% Pyrco-Elo1 pYES2 + SDA, 18:4ω3/17.9% ETA, 20:4ω3/34.1% 65.6%  Pyrco- Elo1pYES2 + EPA, 20:5ω3/2.1% DPA, 22:5ω3/trace — Pyrco- Elo1 pYES2 + ALA,18:3ω3/56.4% ETrA, 20:3ω3/0.3% 0.5% Pyrco- Elo2 pYES2 + SDA,18:4ω3/51.7% ETA, 20:4ω3/0.7% 1.3% Pyrco- Elo2 pYES2 + EPA, 20:5ω3/0.6%DPA, 22:5ω3/1.8% 75.0%  Pyrco- Elo2Isolation and Characterisation of a Pyramimonas CS-0140 Δ6-elongaseIsolation of a Pyramimonas CS-0140 Δ6-elongase Gene Fragment

From an alignment of elongase amino acid sequences from GenBankaccession numbers AB094747, CA158897, CAJ30869, CAL23339 and AAV67797,we identified the consensus amino acid sequence blocks KIYEFVDT (SEQ IDNO:33) and VHVCMYT (SEQ ID NO:34) corresponding to amino acid positions143-150 and 199-205, respectively, of AAV67797. The degenerate primers5′-AARATMTAYGAGTTYGTIGATAC-3′ (SEQ ID NO:50) and5′-TAIGTGTACATGCACACRTGWACCC-3′ (SEQ ID NO:51) (abbreviations as above)were synthesised based on the sequences of these two blocks. An RT-PCRamplification was carried out using the Superscript III One-Step RT-PCRsystem with 100 ng of Pyramimonas CS-0140 total RNA. A 191 bp ampliconwas generated, ligated into pGEM-T Easy and sequenced.

Isolation of a Full Length Pyramimonas CS-0140 Δ6-elongase Gene

Primers were designed to extend the 191 bp fragment by 5′- and 3′-RACE.The 3′ end of the gene was isolated using the gene specific forwardprimer 5′-TTCGTGGATACGTTCATCATGC-3′ (SEQ ID NO:52) as described inExample 1. A 945 bp amplicon was generated, ligated into pGEM-T Easy andsequenced. The 5′ end of the gene was isolated from 1 μg of PyramimonasCS-0140 total RNA using the GeneRacer Kit with a reverse-transcriptionincubation of 55° C. for 1 hour to generate 5′-adapted cDNA as describedin the accompanying manual. The GeneRacer 5′ Primer and the genespecific reverse primer 5′-AGTTGAGCGCCGCCGAGAAGTAC-3′ (SEQ ID NO:53)were used in a PCR amplification using PFU Ultra II Fusion DNApolymerase. This product was then diluted 1:10 and 1 μl used as templatein a second round of PCR using the GeneRacer 5′ Nested Primer5′-GGACACTGACATGGACTGAAGGAGTA-3′ (SEQ ID NO:47) and the gene specificreverse primer 5′-ACCTGGTTGACGTTGCCCTTCA-3′ (SEQ ID NO:54) using thesame PCR conditions as used in the first round of amplification. A 743bp amplicon was generated, ligated into pGEM-T Easy and sequenced. Thethree partial sequences were then assembled into one predicted fulllength sequence.

The full length coding region with a short region of 5′ UTR was thenamplified from total RNA by RT-PCR. The forward primer5′-GCTATGGAGTTCGCTCAGCCT-3′ (SEQ ID NO:55) and the reverse primer5′-TTACTACTGCTTCTTGCTGGCCAGCT-3′ (SEQ ID NO:56) were used with 100 ng ofPyramimonas CS-0140 total RNA. A 900 bp amplicon generated, ligated intopGEM-T Easy and sequenced. The nucleotide sequence of the open readingframe of the amplicon is given as SEQ ID NO:3 and the amino acidsequence of the encoded protein is given as SEQ ID NO:4.

BLAST analysis indicated that the full-length amino acid sequenceprovided as SEQ ID NO:4) has similarity to other Δ5- and Δ6-elongases.The maximum degree of identity between the Pyramimonas CS-0140 elongaseand other proteins (BLASTX) was 54% with AAV67797, the Ostreococcustauri polyunsaturated fatty acid elongase 1. A sequence relationshiptree based on multiple alignment of sequences similar to the PyramimonasCS-0140 elongase, including those used to design the original degenerateprimers, is provided in FIG. 4. The conserved GNS1/SUR4 family domain(NCBI conserved domain pfam01151) is represented in this sequence atamino acids 52 to 297, which typically indicates that the protein isinvolved in long chain fatty acid elongation systems.

Function Characterisation of the Pyramimonas CS-0140 Δ6-elongase inYeast

The entire protein coding region of this clone, contained within anEcoRI fragment in pGEM-T Easy was inserted into pYES2 at the EcoRI site,generating the vector pYES2+Pyrco-Elo1 for introduction and functionalcharacterisation in yeast. Cells of yeast strain INVSC1 were transformedwith pYES2+Pyrco-Elo1 and transformants were selected on medium withouturacil. The yeast cells containing pYES2+Pyrco-Elo1 were grown inculture and then induced by galactose to express the Pyrco-Elo1 cDNA.Fatty acids were added to the culture medium to a final concentration of0.5 mM and further cultured at 30° C. for 48 hrs, after which the fattyacids in total cellular lipids were analysed. When ALA was added to themedium, the presence of ETrA in the cellular lipid of the yeasttransformants was detected at 5.3% of total fatty acids, representing aconversion efficiency (Δ9-elongase activity) of 9.3%. When SDA was addedto the medium, the presence of ETA in the cellular lipid of the yeasttransformants was detected at 34.1%, representing 65.6% conversionefficiency, a high level of Δ6-elongase activity. However, when EPA wasadded to the medium, the presence of DPA in the cellular lipid of theyeast transformants was not detected (Table 5), indicating the cDNAencoded Δ6-elongase activity with some Δ9-elongase activity, but noΔ5-elongase activity in the yeast cells.

The data described above for the two Δ6-elongase genes showed that thegene from Pyramimonas encoded an enzyme that was much more active thanthe gene from Micromonas. This was unexpected. The possibilities thatthe coding region amplified from the Micromonas genomic DNA contained amutation or that the coding region was incomplete were not excluded.

Example 3 Isolation and Characterisation of cDNAs Encoding Δ5-elongasefrom Microalgae

Isolation of a Pyramimonas CS-0140 Δ5-Elongase Gene Fragment

The Pyramimonas CS-0140 strain in the CSIRO Living Collection ofMicroalgae was identified as a micro algal strain that had a high nativelevel of Δ5- and Δ6-elongation (Table 4).

An alignment was carried out of elongase amino acid sequences fromGenBank accession numbers AAV67798 and AB098084. From numerous matchingsequences, we chose the consensus amino acid sequence blocks YLELLDT(SEQ ID NO:35) and MYSYY (SEQ ID NO:36) corresponding to amino acidpositions 136-142 and 198-202, respectively, of AAV67798. The degenerateprimers 5′-ARTAYYTSGARYTRYTGGAYAC-3′ (SEQ ID NO:57) and5′-CATKARRTARTASGAGTACAT-3′ (SEQ ID NO:58) (abbreviations as above) weresynthesised based on the sequences of these two blocks. An RT-PCRamplification was carried out using the Superscript III One-Step RT-PCRsystem as described in Example 1. 0.5 μl of this reaction was then usedas template in a second round of PCR using Taq DNA polymerase (NEB) withthe same primers. A 200 bp amplicon was generated, ligated into pGEM-TEasy and sequenced.

Isolation of a Full Length Pyramimonas CS-0140 Δ5-elongase Gene

Primers were designed to extend the 200 bp fragment by 5′- and 3′-RACE.The 3′ end of the gene was isolated using the gene specific forwardprimer 5′-CATCATACCCTGTTGATCTGGTC-3′ (SEQ ID NO:59) and an oligo-dTreverse primer as in Example 1. A 408 bp amplicon was generated, ligatedinto pGEM-T Easy (Promega) and sequenced. The 5′ end of the gene wasisolated from 1 μg of Pyramimonas CS-0140 total RNA using the GeneRacerKit with a reverse-transcription incubation of 55° C. for 1 hour togenerate 5 ‘-adapted cDNA as described in the accompanying manual. Thegene specific reverse primer 5’-CCAGATCAACAGGGTATGATGGT-3′ (SEQ IDNO:60) was used in the PCR amplification using PFU Ultra II Fusion DNApolymerase as specified by the manufacturer. This product was thendiluted 1:10 and 1 μl used as template in a second round of PCR usingthe GeneRacer 5′ Nested Primer 5′-GGACACTGACATGGACTGAAGGAGTA-3′ (SEQ IDNO:47) and the gene specific reverse primer5′-CGAAAGCTGGTCAAACTTCTTGCGCAT-3′ (SEQ ID NO:61). A 514 bp amplicon wasgenerated, ligated into pGEM-T Easy (Promega) and sequenced. The fulllength sequence was assembled from the three partial sequences.

The full length coding region with a short region of 5′ UTR was thenamplified from total RNA by RT-PCR. The forward primer5′-AACATGGCGTCTATTGCGATTCCGGCT-3′ (SEQ ID NO:62) and the reverse primer5′-TTATTACTGCTTCTTGGCACCCTTGCT-3′ (SEQ ID NO:63) were used in a RT-PCRamplification as described in Example 1. An 810 bp amplicon wasgenerated, ligated into pGEM-T Easy and sequenced. The nucleotidesequence of the open reading frame of the insert as provided as SEQ IDNO:5, and the predicted amino acid sequence encoded by the cDNA is shownas SEQ ID NO:6.

BLAST analysis indicated that the full-length amino acid sequence hadhomology with other Δ5- and Δ6-elongases. BLASTP analysis showed thatthe maximum degree of identity between the Pyramimonas CS-0140 elongaseand other proteins in the Genbank database was 46%, with Accession No.ABR67690 corresponding to a Pavlova viridis C20 elongase. A sequencerelationship tree based on multiple alignment of sequences similar tothe Pyramimonas CS-0140 elongase, including those used to design theoriginal degenerate primers, is provided in FIG. 5.

Functional Characterisation of the Pyramimonas CS-0140 Δ5-elongase inYeast

The entire protein coding region of this clone, contained within anEcoRI fragment of the cDNA in pGEM-T Easy was inserted into pYES2 at theEcoRI site, generating pYES2+Pyrco-Elo2 for introduction and functionalcharacterisation in yeast. Cells of yeast strain INVSC1 were transformedwith pYES2+Pyrco-Elo2 and transformants were selected on medium withouturacil. The yeast cells containing pYES2+Pyrco-Elo2 were grown inculture and then induced by galactose to express the cDNA. After theaddition of fatty acids to the culture medium and 48 hours of furtherculturing at 30° C., the fatty acids in cellular lipids were analysed.When ALA was added to the medium the presence of ETrA in the cellularlipid of the yeast transformants was detected at 0.3% of total fattyacids, representing an 0.5% conversion efficiency (Δ9-elongaseactivity). When SDA was added to the medium the presence of ETA in thecellular lipid of the yeast transformants was detected at 0.7%,representing a 1.3% conversion efficiency (Δ6-elongase activity). WhenEPA was added to the medium, the presence of DPA in the cellular lipidof the yeast transformants was detected at 1.8%, representing asurprisingly high 75% conversion efficiency, indicating strongΔ5-elongase activity in the yeast cells (Table 6).

The present inventors believe such efficient conversion of EPA to DPA ina recombinant cell has not been reported previously. It is predictedthat the conversion efficiency in planta for this enzyme will besimilarly high. The conserved GNS1/SUR4 family domain (NCBI conserveddomain pfam01151) is represented in this sequence at amino acids 50 to267, which typically indicates that the protein is involved in longchain fatty acid elongation systems.

TABLE 6 Conversion of fatty acids in yeast cells transformed withgenetic constructs expressing elongases isolated from Micromonas CS-0170and Pyramimonas CS-0140. Fatty acid precursor/ Fatty acid formed/Conversion Clone % of total FA % of total FA ratio pYES2 + ALA,18:3ω3/52.2% ETrA, 20:3ω3/0.2% 0.4% Mic-Elo1 pYES2 + SDA, 18:4ω3/54.3%ETA, 20:4ω3/0.2% 0.4% Mic-Elo1 pYES2 + EPA, 20:5ω3/2.0% DPA, 22:5ω3/0%  0% Mic-Elo1 pYES2 + ALA, 18:3ω3/51.4% ETrA, 20:3ω3/5.3% 9.3%Pyrco-Elo1 pYES2 + SDA, 18:4ω3/17.9% ETA, 20:4ω3/34.1% 65.6%  Pyrco-Elo1pYES2 + EPA, 20:5ω3/2.1% DPA, 22:5ω3/trace — Pyrco-Elo1 pYES2 + ALA,18:3ω3/56.4% ETrA, 20:3ω3/0.3% 0.5% Pyrco-Elo2 pYES2 + SDA, 18:4ω3/51.7%ETA, 20:4ω3/0.7% 1.3% Pyrco-Elo2 pYES2 + EPA, 20:5ω3/0.6% DPA,22:5ω3/1.8% 75.0%  Pyrco-Elo2

Example 4 Isolation and Characterisation of Genes Encoding Δ6-desaturasefrom Microalgae

Synthesis of a Full Length Micromonas CCMP1545 Δ6-desaturase Gene

The Micromonas CCMP1545 filtered protein models genome sequence producedby the US Department of Energy Joint Genome Institute(http://www.jgi.doe.gov/) was analysed with the BLASTP program using theOstreococcus tauri Δ6-desaturase amino acid sequence, Genbank AccessionNo. AAW70159, as the query sequence. This analysis revealed the presenceof a predicted protein in Micromonas CCMP1545 that had homology withAAW70159. The Micromonas CCMP1545 predicted protein sequence was used todesign and synthesize a codon-optimized nucleotide sequence that wasmost suitable for expression in dicotyledonous plants such as Brassicanapus. The nucleotide sequence of the protein coding region is given inSEQ ID NO:7. The plasmid construct was designated pGA4. The amino acidsequence is shown as SEQ ID NO:8.

BLASTP analysis using the Micromonas CCMP1545 desaturase amino acidsequence SEQ ID NO:8 as query to other proteins in the Genbank databaseshowed that the protein had homology with Δ6-desaturases. The highestdegree of identity was 66% along the full-length with the amino acidsequence of Accession No. AAW70159, the sequence of an Ostreococcustauri Δ6-desaturase. A sequence relationship tree based on multiplealignment of sequences similar to the Micromonas CCMP1545 desaturase isprovided in FIG. 6. This front-end desaturase contains a cytochrome b5domain (NCBI conserved domain pfam00173) at amino acids 54 to 104 andthe Δ6-FADS-like conserved domain (NCBI conserved domain cd03506) atamino acids 172 to 428. The three histidine boxes indicative of afront-end desaturase are present in this sequence at 190-195, 227-232and 401-405, respectively. Proteins containing both of these domains aretypically front-end desaturases required for the synthesis of highlyunsaturated fatty acids. Interestingly, this desaturase clusters closelywith AAW70159, the only biochemically confirmed plant-like acyl-CoAdesaturase published to date.

Function Characterisation of the Micromonas CCMP1545 Δ6-desaturase inYeast Cells

The entire coding region of the Micromonas desaturase, contained withina KpnI-SacI fragment from plasmid pGA4 was inserted into yeast vectorpYES2 at the KpnI-SacI site, generating pYES2+Micd6D for introductionand functional characterisation in yeast. Cells of yeast strain INVSC1were transformed with pYES2+Micd6D and transformants were selected onmedium without uracil. The yeast cells containing pYES2+Micd6D weregrown in culture and then induced by galactose. After the addition of0.5 mM LA, ALA, ETrA, DGLA or ETA to the culture medium and 48 hours offurther culturing at 30° C., the fatty acids in total cellular lipidswere analysed. When LA was added to the medium the presence of GLA inthe cellular lipid of the yeast transformants was detected at 3.9% oftotal fatty acids, representing a Δ6-desaturation conversion efficiencyof 11.4%. When ALA was added to the medium the presence of SDA in thecellular lipid of the yeast transformants was detected at 13.9% of totalfatty acids, representing a Δ6-desaturation conversion efficiency of39.0%. That is, the conversion efficiency for ω3 fatty acid substrateswas 3.5-fold greater than for the corresponding ω6 fatty acid substrate.When ETrA was added to the medium the presence of ETA in the cellularlipid of the yeast transformants was detected at 0.21% of total fattyacids, representing a Δ8-desaturation conversion efficiency of 8.0%.However, when either DGLA or ETA were added to the medium, the presenceof ARA or EPA, respectively, was not detected. This indicated theabsence of any Δ5-desaturation activity (Table 7).

TABLE 7 Conversion of fatty acids in yeast cells transformed withgenetic constructs expressing desaturases isolated from MicromonasCCMP1545, Ostreococcus lucimarinus and Pyramimonas CS-0140. Fatty acidprecursor/ Fatty acid formed/ Conversion Clone % of total FA % of totalFA ratio pYES2 + LA, 18:2ω6/30.3% GLA, 18:3ω6/3.9% 11.4%  Mic-d6DpYES2 + ALA, 18:3ω3/21.7% SDA, 18:4ω3/13.9% 39.0%  Mic-d6D pYES2 + ETrA,20:3ω3/2.4% ETA, 20:4ω3/0.21% 8.0% Mic-d6D pYES2 + DGLA, 20:3ω6/2.6%ARA, 20:4ω6/0% — Mic-d6D pYES2 + ETA, 20:4ω3/6.2% EPA, 20:5ω3/0% —Mic-d6D pYES2 + LA, 18:2ω6/29.5% GLA, 18:3ω6/2.1% 6.6% Ostlu-d6D pYES2 +ALA, 18:3ω3/21.8% SDA, 18:4ω3/13.8% 38.8%  Ostlu-d6D pYES2 + ETrA,20:3ω3/2.2% ETA, 20:4ω3/0% — Ostlu-d6D pYES2 + GLA, 18:3ω6/29.2%18:4ω6/0% — Ostlu-d6D pYES2 + SDA, 18:4ω3/41.7% 18:5ω3/0% — Ostlu-d6DpYES2 + DGLA, 20:3ω6/2.3% ARA, 20:4ω6/0% — Ostlu-d6D pYES2 + ETA,20:4ω3/4.9% EPA, 20:5ω3/0% — Ostlu-d6D pYES2 + LA, 18:2ω6/35.1% GLA,18:3ω6/0% — Pyrco-d5D pYES2 + ALA, 18:3ω3/40.9% SDA, 18:4ω3/0% —Pyrco-d5D pYES2 + DGLA, 20:3ω6/2.9% ARA, 20:4ω6/0.12% 4.0% Pyrco-d5DpYES2 + ETA, 20:4ω3/7.2% EPA, 20:5ω3/0.26% 3.5% Pyrco-d5DFunction Characterisation of the Micromonas CCMP1545 Δ6-desaturase inPlant Cells

The enzyme activities of the Micromonas CCMP1545 Δ6-desaturase(Mic1545-d6D) and an Echium plantagineum Δ6-desaturase (Echp1-d6D; Zhouet al., 2006), used here as a positive control sample, were demonstratedin planta using an enhanced Nicotiana benthamiana transient expressionsystem as described in Example 1. A vector designated 35S-pORE04 wasmade by inserting a PstI fragment containing a 35S promoter into theSfoI site of vector pORE04 after T4 DNA polymerase treatment to bluntthe ends (Coutu et al., 2007). A genetic construct 35S:Mic1545-d6D wasmade by inserting the entire coding region of pGA4, contained within aSwaI fragment, into 35S-pORE04 at the SmaI-EcoRV site, generatingpJP2064.

These chimeric vectors were introduced into Agrobacterium tumefaciensstrain AGL1 and cells from cultures of these infiltrated into leaftissue of Nicotiana benthamiana plants in the greenhouse. The plantswere grown for a further five days after infiltration before leaf discswere taken for GC analysis which revealed that both genes werefunctioning as Δ6-desaturases in Nicotiana benthamiana.

Leaf tissue transformed with the Echium plantagineum Δ6-desaturasecontained GLA (0.4%) and SDA (1.2%), which represented conversionefficiencies of 3.8% and 4.4%, respectively. Leaf tissue transformedwith the Micromonas CCMP1545 Δ6-desaturase contained SDA (2.2%) whichrepresented a conversion efficiency of 6.9% but no detectable GLA. Theabsence of GLA in the leaf tissue could be due to an extreme preferencein planta for the ω3 substrate ALA compared with the ω6 substrate LA, orin part to the presence of native Nicotiana benthamiana ω3 desaturaseactivity which would convert some of the GLA produced by Δ6-desaturationto SDA. Such effects have been noted as likely in previous experimentsdescribing acyl-PC Δ6-desaturases with ω3 substrate preference (Sayanovaet al., 2006), although the extent to which this occurs was notquantified in that study.

Omega-3 Substrate Preference of Micromonas Δ6-desaturase

The Δ6-desaturase isolated from Micromonas had a surprisingly strongpreference for ω3 substrates in planta as well as in yeast. The enzymeexpressed in yeast cells was observed to have 3.5-fold greater activityon ω3-desaturated fatty acid substrates than the correspondingω6-desaturated fatty acid substrates. The observed preference for ω3substrates was entirely surprising and unexpected based on the reportedlack of preference for the O. tauri enzyme (Domergue et al., 2005).Reports on expression of the O. tauri Δ6-desaturase in yeast or in plantseed indicate similar activity on LA and ALA.

The use of this gene or other genes with such high specificity forω3-desaturated fatty acid substrates together with other fatty aciddesaturases and elongases as part of a recombinant VLC-PUFA pathway inplants was therefore predicted to increase the levels of EPA, DPA andDHA relative to the use of desaturases without preference forω3-desaturated substrates. Such an increase was predicted to occur as aresult of reducing the conversion of LA to GLA and the subsequent ω6PUFAs DGLA and ARA which are not efficiently converted in planta totheir ω3 counterparts by fungal or yeast Δ17-desaturases. Whilst a Δ6desaturase with a preference for ω3 fatty acid substrates has beenisolated (Sayanova et al., 2003), it had activity on phospholipid-linkedacyl chains. In contrast, the desaturase obtained from Micromonas arepredicted to have activity on acyl-CoA substrates.

Dual Δ6/Δ8 Function of Micromonas CCMP1545 Δ6-desaturase

It was interesting to note that the Micromonas CCMP1545 Δ6-desaturasedisplayed a significant level of Δ8-desaturase activity and so hadsignificant dual activities, in contrast to the Ostreococcus lucimarinusenzyme which did not have detectable Δ8-desaturase activity (below). Thedual desaturase activity is predicted to be useful in the constructionof dual Δ6/Δ8-desaturase pathways in planta, or where the elongase thatis used in construction of such pathways has both Δ9-elongase andΔ6-elongase activities. The use of such a gene would help to reduce theaccumulation of ETrA by converting it to ETA, which would then beΔ5-desaturated to EPA.

Synthesis of a Full Length Ostreococcus lucimarinus Δ6-desaturase Gene

The GenBank database of non-redundant protein sequences was analysed byBLASTX using the Ostreococcus tauri Δ6-desaturase nucleotide sequence(Accession No. AY746357) as the query sequence. From this analysis, anOstreococcus lucimarinus gene was identified which encoded apartial-length protein with amino acid sequence of Accession No.XP_001421073. The genomic DNA sequences flanking the region coding forXP_001421073 were then examined to identify putative translation startand stop codons to define the full-length protein coding region, thenucleotide sequence of which is given as SEQ ID NO:9. The coding regionwas then translated into a protein sequence, given as SEQ ID NO:10. Thisamino acid sequence was used to design and synthesize a codon-optimizednucleotide sequence that was most suitable for expression in Brassicanapus and other dicotyledonous plants, having the nucleotide sequenceshown in SEQ ID NO:11.

BLASTP analysis using the Ostreococcus lucimarinus desaturase amino acidsequence as query to other proteins in the Genbank database showed thatSEQ ID NO:10 had homology with Δ6-desaturases. The highest degree ofidentity along the full-length sequence was 76% with the amino acidsequence of Accession No. AAW70159, the sequence for the Ostreococcustauri Δ6-desaturase. A sequence relationship tree based on multiplealignment of sequences similar to the Ostreococcus lucimarinusdesaturase is provided in FIG. 7. This front-end desaturase contained acytochrome b5 domain (NCBI conserved domain pfam00173) at amino acids 55to 108 and the Δ6-FADS-like conserved domain (NCBI conserved domaincd03506) at amino acids 198 to 444. The three histidine boxes indicativeof a front-end desaturase are present in this sequence at amino acids207-212, 244-249 and 417-421. Proteins containing both of these domainsare typically front-end desaturases required for the synthesis of highlyunsaturated fatty acids. Interestingly, this desaturase clusters closelywith AAW70159, the only biochemically confirmed plant-like acyl-CoAdesaturase published to date.

Functional Characterisation of the Ostreococcus lucimarinusΔ6-desaturase in Yeast Cells

The entire coding region of the Ostreococcus gene (SEQ ID NO:11),contained within a NotI fragment in pGEM-T Easy was inserted into pYES2at the NotI site, generating the chimeric vector pYES2+Ostlud6D, forintroduction and functional characterisation in yeast. Cells of yeaststrain INVSC1 were transformed with pYES2+Ostlud6D and transformantswere selected on medium without uracil. The yeast cells containingpYES2+Ostlud6D were grown in culture and then induced by galactose.After the addition of LA, ALA, SDA or EPA each to a final concentrationof 0.5 mM in the culture medium, and 48 hours of further culturing at30° C., the fatty acids in cellular lipids were analysed. When substrateLA was added to the medium, the presence of product GLA in the cellularlipid of the yeast transformants was detected at 2.1% of total fattyacids, representing a Δ6-desaturation conversion efficiency of 6.6%.When substrate ALA was added to the medium, the presence of product SDAwas detected at 13.8% of total fatty acids in the cellular lipid of theyeast transformants, representing a Δ6-desaturation conversionefficiency of 38.8%. However, when any of ETrA, DGLA or ETA were addedto the medium, the presence of ETA, ARA or EPA, respectively, was notdetected. This indicated the absence of any Δ5- or Δ8-desaturationactivity (Table 7), and also a preference for the ω3 fatty acidsubstrate relative to the corresponding ω6 fatty acid of the same lengthand unsaturation pattern.

Acyl-CoA Substrate Specificity of Desaturases

The desaturases described in this Example were more closely related tothe previously isolated Δ6-desaturase from Ostreococcus tauri than toother Δ6-desaturases (FIG. 9). This similarity was further highlightedwhen a phylogenetic tree of these genes alongside other members of thedesaturase family was produced (FIG. 10). The Ostreococcus tauriΔ6-desaturase has been reported to be active on acyl-CoA substrates(Domergue et al., 2005). Based on these observations, it was predictedthat the Δ6-desaturases encoded by the genes described above would alsobe active on acyl-CoA substrates rather than acyl-PC substrates.Interestingly, the Pavlova salina Δ5-desaturase also clustered with theO. tauri Δ6-desaturase and the Δ8-desaturase formed a separate branch.

To establish whether the M. pusilla (Micromonas CCMP1545) Δ6-desaturaseis capable of using acyl-CoA fatty acids as substrates and therebyproducing Δ6-desaturated acyl-CoA fatty acids, S. cerevisiae wastransformed with a gene construct encoding the desaturase alone andtriplicate cultures of the transformant cell lines grown in the presenceof 250 μM exogenous 18:3^(Δ9,12,15). Total lipids were then extractedfrom the cultures and fractionated into neutral lipids (NL),phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine(PS) and phosphatidylethanolamine (PE) classes by thin layerchromatography (TLC), after which FAME were produced from each class andanalysed by GC. The data is shown in Table 8.

TABLE 8 Fatty acid composition (percent of total fatty acids) of totallipid and fractionated neutral lipids (NL), phosphatidylcholine (PC),phosphatidylinositol (PI), phosphatidylserine (PS) andphosphatidylethanolamine (PE) of S. cerevisiae transformed with the M.pusilla Δ6-desaturase cloned into pYES2. Fatty acid Total NL PC PI PS PE16:0 24.3 ± 1.5 24.5 ± 4.3 36.8 ± 3.4 45.6 ± 2.4 45.8 ± 3.9 37.0 ± 3.916:1^(Δ3t) 14.7 ± 0.4 12.1 ± 0.9 15.7 ± 0.5  8.1 ± 0.7 19.4 ± 1.0 21.5 ±3.5 18:0  8.1 ± 0.7 11.5 ± 1.4 11.7 ± 0.7 17.4 ± 3.1  0.9 ± 1.3  3.6 ±3.8 18:1^(Δ9) 11.0 ± 1.3 10.0 ± 2.0  7.0 ± 1.2 14.6 ± 0.9 26.3 ± 1.818.6 ± 2.5 18:3^(Δ9,12,15) 12.1 ± 0.7 12.2 ± 2.4  7.1 ± 0.8  5.6 ± 0.1 2.3 ± 2.0  6.9 ± 2.3 18:4^(Δ6,9,12,15) 29.4 ± 0.9 29.4 ± 1.9 21.0 ± 0.2 7.7 ± 0.7  4.1 ± 0.3  9.9 ± 1.9 Other 0.3 0.3 0.8 1.1 1.2 2.5 Total 100100 100 100 100 100

In the total lipids, 71% of 18:3^(Δ9,12,15) had been Δ6-desaturated to18:4^(Δ6,9,12,15). No enrichment of the product in the PC fraction wasdetected when compared with the total lipid extract. Indeed, there was asubstantially lower percentage of 18:4^(Δ6,9,12,15) in the PC fractionthan in the total lipids (21.0% vs. 29.4%), indicating that thedesaturase was producing 18:4^(Δ6,9,12,15) as an acyl-CoA thioester(Domergue et al., 2003).

The gene encoding the M. pusilla Δ6-desaturase was also introduced intoArabidopsis plants by transformation. A genetic constructLinin:Micpu-d6D was generated by inserting the entire coding region ofthe M. pusilla Δ6-desaturase, contained within a SwaI fragment, intoLinin-pWVEC8 at the SmaI site, generating linP-mic1545-d6D-linT. Thepromoter for this construct was the seed-specific linin promoter fromflax. This construct was transformed into A. thaliana ecotype Columbiaand the fatty acid composition in T2 seeds of the transformed plantsanalysed by GC (FIG. 11).

Biochemical studies in both yeast and N. benthamiana provided evidencethat the Δ6-desaturase from M. pusilla is an acyl-CoA desaturase.Analysis of the kinetics of an ensuing elongation step has been used inother studies as an indirect method to determine the ability of adesaturase to yield an acyl-CoA product: the availability of theΔ6-desaturated product (SDA) for the subsequent Δ6-elongation step,which occurs in the acyl-CoA metabolic pool, is affected by thesubstrate specificity of the Δ6-desaturase (Domergue et al., 2003, 2005;Hoffmann et al., 2008). Similar rates of Δ6-elongation were obtainedwhen the Δ6-desaturases from O. tauri and M. pusilla were used, incontrast with the significantly lower level of elongation observed whenthe E. plantagineum acyl-PC Δ6-desaturase was used (FIG. 12a ). Furtherevidence was observed when the distribution of the Δ6-desaturase productSDA in the yeast lipid classes was analysed (Table 8). No enrichment inthe PC fraction was observed when compared with the total lipid fractionalthough such enrichment would be expected if and when the SDA wasproduced by an acyl-PC desaturase (Domergue et al., 2005). Therelatively low levels of Δ6-desaturation observed in our study (FIG. 12)were expected since the bulk of the substrates LA and ALA in N.benthamiana leaf are located in the plastid and are unavailable fordesaturation. However, since these fatty acids are also isolated duringFAME preparation their presence effectively reduces the calculatedoverall conversion efficiency. Seed-specific conversion efficiencieswould therefore be expected to be much higher with the same genes.

Comparison Between Acyl-CoA and Acyl-PC Δ6-desaturases

Additional comparisons were made between the Micromonas CCMP1545Δ6-desaturase, Echium plantagineum Δ6-desaturase and Ostreococcus tauriΔ6-desaturase (Domergue et al., 2005) in plant cells. Genetic constructs35S:Mic1545-d6D and 35S:Echp1-d6D as described in Example 4 werecompared with a genetic construct 35S:Ostta-d6D which was made byinserting the entire coding region of the Ostreococcus tauriΔ6-desaturase, contained within a SwaI fragment, into 35S-pORE04 at theSmaI-EcoRV site, generating pJP3065.

Direct comparisons between the E. plantagineum and either the O. tauriand M. pusilla EPA pathways showed that the acyl-CoA desaturase pathwaysyielded far higher levels of EPA due to both more efficientΔ6-desaturation and more efficient, subsequent Δ6-elongation (FIG. 12a). The E. plantagineum Δ6-desaturase catalysed conversion of 14% of theω3 substrate (18:3^(Δ9,12,15) to 18:4^(Δ6,9,12,15)) and 30% of the ω6substrate (18:2^(Δ9,12) to 18:3^(Δ6,9,12)). Use of the O. tauriΔ6-desaturase resulted in 24% ω3 conversion and 40% ω6 conversion whilstuse of the M. pusilla Δ6-desaturase resulted in 27% ω3 conversion and15% ω6 conversion. These conversions resulted in the production of 1.3%20:4^(Δ5,8,11,14) and 3.4% 20:5^(Δ5,8,11,14,17) for the E. plantagineumpathway, 1.2% 20:4^(Δ5,8,11,14) and 9.6% 20:5^(Δ5,8,11,14,17) for the O.tauri pathway and 0.6% 20:4^(Δ5,8,11,14) and 10.7% 20:5^(Δ5,8,11,14,17)for the M. pusilla pathway.

Δ6-elongation was far higher when either the O. tauri or M. pusillaΔ6-desaturases produced the substrate 18:4^(Δ6,9,12,15) compared to whenthe E. plantagineum desaturase was used (FIG. 12a ). In addition to theω3 substrate specificity shown by the M. pusilla Δ6-desaturase, the P.cordata Δ6-elongase (see Example 2) proved to be highly specific andconverted the ω3 substrate 18:4^(Δ6,9,12,15) at a far higher rate than18:3^(Δ6,9,12) (89% and 21%, respectively, for the M. pusilla EPApathway).

Use of Dual Δ6-desaturase Pathways

Comparisons were made in which the possibility of increasingΔ6-desaturation by using a pathway containing two Δ6-desaturases wasexplored. First, the combination of the E. plantagineum acyl-PCdesaturase and the M. pusilla acyl-CoA desaturase did not significantlyincrease conversion efficiencies above those seen in a pathwaycontaining only the M. pusilla desaturase (FIG. 12b ). Similar resultswere obtained when the E. plantagineum and the O. tauri Δ6-desaturaseswere combined. A dual acyl-CoA Δ6-desaturase pathway in which both theO. tauri and M. pusilla desaturases were combined also did not result inincreased ω3 conversion efficiencies when compared with either the O.tauri or M. pusilla pathways (FIG. 12c ).

The effect of using dual Δ6-desaturases in an EPA-producing pathway wasalso tested. The first test was to combine the acyl-PC desaturase fromE. plantagineum with both of the acyl-CoA desaturases in separateexperiments. It was hypothesised that the addition of a lipid-linkeddesaturase might increase the conversion of any acyl-PC substrate LA orALA to GLA or SDA, respectively. Similarly, we also tested whether theuse of two acyl-CoA desaturases might increase the accumulation of EPA.Neither of these scenarios proved true in the transient assays in N.benthamiana.

Example 5 Isolation and Characterisation of Genes Encoding Δ5-desaturasefrom Microalgae

Isolation of a Pyramimonas CS-0140 Δ5-Desaturase Gene Fragment

An alignment of desaturase amino acid sequences from GenBank accessionnumbers ABL96295, ABP49078, XP_001421073, AAM09687, AAT85661, AAW70159and AAX14505 identified the consensus amino acid sequence blocksWKNMHNKHHA (SEQ ID NO:37) and HHLFPSMP (SEQ ID NO:38) corresponding toamino acid positions 197-206 and 368-375, respectively, of ABL96295. Thedegenerate primers 5′-GGTGGAAGAACAAGCACAACrdncaycaygc-3′ (SEQ ID NO:64)and 5′-GGGCATCGTGGGGwanarrtgrtg-3′ (SEQ ID NO:65) were designed usingthe CODEHOP program (Rose et al., 1998) based on the sequences of thesetwo blocks. A touchdown PCR amplification was carried out using Taq DNApolymerase (NEB) in a volume of 20 μL using 10 pmol of each primer, 50ng of Pyramimonas CS-0140 genomic DNA with buffer and nucleotidecomponents as specified in the accompanying manual. The cyclingconditions were: 1 cycle of 94° C. for 3 minutes; 20 cycles of 94° C.for 1 minute, 70° C. for 2 minutes (−1° C. per cycle), 72° C. for 1minute; 20 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C.for 1 minute; 1 cycle of 72° C. for 5 minutes; 4° C. hold. A 551 bpamplicon was generated, ligated into pGEM-T Easy (Promega) andsequenced.

Isolation of a Full Length Pyramimonas CS-0140 Δ5-desaturase Gene

Primers were designed to extend the 551 bp fragment by 5′- and 3′-RACEand used as described in Example 1. The 3′ end of the cDNA for the geneencoding the Δ5-desaturase was isolated using the gene specific forwardprimer 5′-AGCGAGTACCTGCATTGGGT-3′ (SEQ ID NO:66) and the modifiedoligo-dT reverse primer as in Example 1. A 477 bp amplicon wasgenerated, ligated into pGEM-T Easy and sequenced. The 5′ end of thegene was isolated by the modified terminal-transferase method as inExample 1. The gene specific reverse primer was5′-ATAGTGCTTGGTGCGCAAGCTGTGCCT-3′ (SEQ ID NO:67). After two rounds ofPCR amplification, a 317 bp amplicon was generated, ligated into pGEM-TEasy (Promega) and sequenced. The three partial sequences were assembledinto the predicted sequence of the full length gene.

The full length protein coding region with a short region of 5′ UTR wasthen amplified from genomic DNA. The forward primer5′-CACCATGGGAAAGGGAGGCAATGCT-3′ (SEQ ID NO:68) and the reverse primer5′-TTACTAGTGCGCCTTGGAGTGAGAT-3′ (SEQ ID NO:69) were used in a PCRamplification using PFU Ultra II Fusion DNA polymerase (Stratagene) in avolume of 20 μL using 4 pmol of each primer and 50 ng of PyramimonasCS-0140 genomic DNA with buffer components as specified in theaccompanying PFU Ultra II Fusion manual. An 1336 bp ampliconrepresenting the full-length cDNA was generated, ligated into pGEM-TEasy and sequenced. The nucleotide sequence of the open reading frame ofthe cDNA is given in SEQ ID NO:12.

BLAST analysis showed that the full-length amino acid sequence encodedby the gene, given as SEQ ID NO:13, encoded a protein with similarity toknown Δ5- or Δ6-desaturases. These two types of desaturases are similarat the amino acid level and it was uncertain from amino acid sequencealone which activity was encoded. Analysis of enzyme activity wascarried out as described below, showing the encoded protein hadΔ5-desaturase activity. The highest degree of identity between thePyramimonas CS-0140 desaturase and other desaturases in the Genbankdatabase as determined by BLASTP was 52%, to Accession No. EDQ92231,which was the amino acid sequence of a protein with undefined enzymeactivity from Monosiga brevicollis MX1. A sequence relationship treebased on multiple alignment of sequences similar to the PyramimonasCS-0140 desaturase, including those used to design the originaldegenerate primers, is provided in FIG. 8. This front-end desaturasecontains a cytochrome b5 domain (NCBI conserved domain pfam00173) atamino acids 16 to 67 and the Δ6-FADS-like conserved domain (NCBIconserved domain cd03506) at amino acids 159 to 411. The three histidineboxes indicative of a front-end desaturase are present in this sequenceat amino acids 175-180, 212-217 and 384-388. Proteins containing thesedomains are typically front-end desaturases required for the synthesisof multiply unsaturated fatty acids.

Function Characterisation of the Pyramimonas CS-0140 Δ5-desaturase inYeast

The entire coding region of this clone, contained within a NotI fragmentin pGEM-T Easy was inserted into pYES2 (Invitrogen) at the NotI site,generating pYES2+Pyrco-des2 for introduction and functionalcharacterisation in yeast. Cells of yeast strain INVSC1 (Invitrogen)were transformed with pYES2+Pyrco-des2 and transformants were selectedon medium without uracil. The yeast cells containing pYES2+Pyrco-des2were grown in culture and then induced by galactose. After the additionof 0.5 mM LA, ALA, DGLA or ETA to the culture medium and 48 hours offurther culturing at 30° C., the fatty acids in cellular lipids wereanalysed. When DGLA was added to the medium, ARA was detected in thecellular lipid of the yeast transformants at 0.12% of total fatty acids,representing a Δ5-desaturation conversion efficiency of 4.0%. When ETAwas added to the medium, EPA was detected in the cellular lipid of theyeast transformants at 0.26% of total fatty acids, representing aΔ6-desaturation conversion efficiency of 3.5%. However, when either LAor ALA were added to the medium, GAL or SDA, respectively, was notproduced in the yeast transformants. This indicated that the protein didnot have any Δ6-desaturation activity in the yeast cells (Table 7).

Expression of the Pyramimonas cordata Δ5-desaturase in Plant Cells

The enzyme activities of the Micromonas CCMP1545 Δ6-desaturase (SEQ IDNO:8 encoded by SEQ ID NO:7), Pyramimonas CS-0140 Δ6-elongase (SEQ IDNO:4 encoded by SEQ ID NO:3) and Pyramimonas CS-0140 Δ5-desaturase (SEQID NO:13 encoded by SEQ ID NO:12) along with the Arabidopsis thalianaDGAT1 (SEQ ID NO:74 encoded by SEQ ID NO:75) were demonstrated in plantausing an enhanced Nicotiana benthamiana transient expression system asdescribed in Example 1.

A genetic construct 35S:Pyrco-d5D encoding the Δ5-desaturase under thecontrol of the constitutive 35S promoter was made by inserting theentire coding region of the Pyramimonas CS-0140 Δ5-desaturase, containedwithin an EcoRI fragment, into 35S-pORE04 (Example 4, above) at theEcoRI site, generating 35S:Pyrco-d5D. The chimeric vectors35S:Mic1545-d6D (Example 10), 35S:Pyrco-d6E (Example 10) and35S:Pyrco-d5D were introduced individually into Agrobacteriumtumefaciens strain AGL1 and transgenic cells from cultures of these weremixed and the mixture infiltrated into leaf tissue of Nicotianabenthamiana plants in the greenhouse. The plants were grown for afurther five days after infiltration before leaf discs were taken for GCanalysis which revealed that these genes were functioning to produce EPAin Nicotiana benthamiana. Leaf tissue transformed with these genescontained SDA (1.0%), ETA (0.1%), EPA (10.0%). The leaf tissue alsocontained trace levels of GLA, ETA and ARA. The Δ5-desaturase conversionefficiency was calculated to be 98.8%.

This experiment demonstrated that the microalgal Δ5-desaturases arecapable of converting ETA to EPA with an efficiency of at least 90% orat least 95% in plant cells.

Example 6 Isolation and Characterisation of Genes Encoding ω3-desaturasefrom Microalgae

Isolation of a Micromonas CS-0170 ω3-desaturase Gene Fragment

In an attempt to determine whether microalgae such as Micromonas hadgenes encoding ω3 desaturases and perhaps identify such a gene, a searchwas made of the Micromonas strain RCC299 genomic sequence for genesshowing homology to FAD3. However, this search failed to identify anycandidate genes. The inventors therefore considered whether an ω3desaturase could be represented in other types of desaturases inMicromonas. This hypothesis was supported by the finding (Example 4)that the Δ6 desaturase in the same strain was of the front-end, acyl-CoAdependent type. However, when examined, the Micromonas RCC299 genomeappeared to contain genes for at least 30 putative fatty aciddesaturases and there was no information as to which of these, if indeedany, might encode an ω3 desaturase.

In one experiment, an alignment of desaturase amino acid sequences fromGenbank accession numbers BAD91495, ABL63813, BAD11952 and AAR20444identified the consensus amino acid sequence blocks WCIGHDCG (SEQ IDNO:39) and TFLQHHDEDM (SEQ ID NO:40) corresponding to amino acidpositions 106-113 and 296-305, respectively, of BAD91495. The degenerateprimers 5′-TGTGGTGCATCGGCCAYGANKSNGG-3′ (SEQ ID NO:70) and5′-TGTCCTCGTCGTTGTGCTGNARRWANGT-3′ (SEQ ID NO:71) were designed usingthe CODEHOP program based on the sequences of these two blocks. Atouchdown PCR amplification was carried out using Taq DNA polymerase(NEB) in a volume of 20 μL using 10 pmol of each primer, 50 ng ofMicromonas CS-0170 genomic DNA with buffer and nucleotide components asspecified in the accompanying manual. The cycling conditions were: 1cycle of 94° C. for 3 minutes; 20 cycles of 94° C. for 1 minute, 70° C.for 2 minutes (−1° C. per cycle), 72° C. for 1 minute; 35 cycles of 94°C. for 1 minute, 56° C. for 1 minute, 72° C. for 1 minute; 1 cycle of72° C. for 5 minutes; 4° C. hold. A 528 bp amplicon was generated,ligated into pGEM-T Easy (Promega) and sequenced. The nucleotidesequence of this amplicon is provided as SEQ ID NO:14, and the encodedpartial protein sequence is provided as SEQ ID NO:15.

Synthesis of a Full Length Micromonas RCC299 ω3-desaturase Gene

The 528 bp fragment generated by degenerate PCR was compared with thecompleted Micromonas RCC299 filtered protein models genome sequence(produced by the US Department of Energy Joint Genome Institutehttp://www.jgi.doe.gov/). BLAST analysis revealed regions of highhomology between a region of Micromonas RCC299 chromosome 13 and SEQ IDNO:14. Based on the near identity of the two sequences it was likelythat the Micromonas strains CS-0170 and RCC299 were very closely related(nucleotide sequence of Micromonas RCC299 provided as SEQ ID NO:16). TheMicromonas RCC299 predicted protein sequence (SEQ ID NO:17) was used todesign and synthesize a codon-optimized nucleotide sequence that wasmost suitable for expression in Brassica napus or other dicotyledonousplants (SEQ ID NO:18). A shorter version of this gene starting atnucleotide of 164 of SEQ ID NO:18 was tested in yeast but no ω3desaturase activity was detected.

BLAST analysis indicated that the full-length amino acid sequence (SEQID NO:17) has homology with FAT-1, FAT-2 and ω3 desaturases. It was notpossible to predict on sequence alone which activity was encoded. Themaximum degree of identity between the Micromonas CS-0170 desaturase andother proteins in the Genbank database by BLASTX was 35% withXP_001899085.1, which was a Brugia malayi protein in the fatty aciddesaturase family. This front-end desaturase contained a Δ12-FADS-likeconserved domain (NCBI conserved domain cd03507). Proteins containingboth of these domains are typically front-end desaturases required forthe synthesis of fatty acids, including the ω3 desaturase family.

Functional Characterisation of the Micromonas RCC299 ω3-desaturase inPlanta

The enzymatic function of the putative ω3-desaturase encoded by thefull-length gene isolated from Micromonas RCC299 (Mic299-ω3D, asdescribed above) and the Phytophthora infestans Δ17-desaturase(Phyin-d17D, GenBank Accession No. CAM55882), used here as a positivecontrol sample, were tested in planta using the enhanced Nicotianabenthamiana transient expression system as described above.

The 35S:Mic299-ω3D construct was built by cloning the entire proteincoding region of SEQ ID NO:18, contained within an EcoRI fragment, intovector 35S-pORE04 (Example 4) at the EcoRI site, generating the geneticconstruct designated pJP2073. The 35S:Phyin-d17D construct was made bycloning the entire coding region of the Phytophthora infestans Δ17desaturase, contained within an EcoRI fragment, into 35S-pORE04 at theEcoRI site, generating pJP2074. Similarly, a 35S:Arath-DGAT1 constructwas built by cloning the entire coding region of the Arabidopsisthaliana DGAT1 (AF051849), contained within an EcoR1 fragment, into35S-pORE04 at the EcoRI site, generating pJP2078.

Agrobacterium tumefaciens strain AGL1 was grown at 28° C. in LB brothsupplemented with 50 mg/mL kanamycin and 50 mg/mL rifampicin tostationary phase. The bacteria were then pelleted by centrifugation at5000 g for 15 min at room temperature before being resuspended toOD600=1.0 in an infiltration buffer containing 10 mM MES pH 5.7, 10 mMMgCl₂ and 100 μM acetosyringone. The cells were then incubated at 28° C.with shaking for 3 hours before equal volumes of Agrobacterium cellscontaining 35S:p19, 35S:Arath-DGAT1 and either 35S:Phyin-d17D or35S:Mic299-ω3D cultures were mixed prior to infiltration into leaftissue. An arachidonic acid salt was prepared and fed to the transformedleaf tissue as described above with leaf discs being taken for analysisat both 5 hours and 24 hours after substrate feeding. Leaf spotsinfiltrated with the 35S:Phyin-d17D construct or, separately, the35S:Mic299-ω3D construct all demonstrated the conversion of ARA (20:4ω6)to EPA, (20:5ω3) at 37% and 50% efficiency, respectively (FIG. 13),indicating that the protein had Δ17-desaturase activity.

Discussion: Characterisation of the First Microalgal ω3-desaturase withΔ7-desaturase Activity

The Micromonas RCC299 ω3 desaturase described in this study is the firstmicroalgal, i.e. plant-like, Δ17 desaturase described, having activityon a C20 or longer fatty acid substrate. Land plants are not known tohave ω3 desaturases of the front-end desaturase type, but rather of theFAD3 type. It was therefore surprising to find that a microalgal strain,which is more related to plants than fungi, possessed an ω3 desaturaseof the front-end desaturase type.

It was considered likely, based on homology to other desaturases, thatthe fungal Phytophthora infestans desaturase used as a control gene inthe experiments described above was active on acyl-PC substrates whilstthe Micromonas RCC299 desaturase was active on acyl-CoA substrates.Other fungal desaturases are known to be active on acyl-PC substrates.This conclusion regarding the Micromonas gene was consistent with itsobserved similarity to the Δ6-desaturase gene from the same strain(Example 4). This substrate preference can be further examined bysubstrate feeding studies where substrates such as ARA fed to thetransformed tissue will be immediately available to the acyl-CoA poolbut available to the acyl-PC pool only after conversion by native plant(e.g. Nicotiana benthamiana) acyltransferases.

The Micromonas RCC299 ω3 desaturase gene will be very useful in theconstruction of recombinant pathways designed to yield EPA anddownstream fatty acids DPA and DHA, and other ω3 VLC-PUFA in plants, inparticular because of its ability to convert ω6 substrates such as ARAto ω3 products. Activity on acyl-CoA substrates enhances this usefulnesswhen combined with elongases such as Δ5-elongases that also operate inthe acyl-CoA pool. Furthermore, the fatty acid profile of Micromonasstrains indicated that the Micromonas enzyme may also have thecapability to convert ω6 C18 fatty acids such as GLA or LA to their ω3counterparts such as SDA or ALA, respectively. Conversion of GLA to SDAcan be demonstrated in either yeast cells or in planta by substratefeeding as described above for substrate ARA, while conversion of LA toALA is better demonstrated in yeast cells because of the presence ofendogenous Δ15 desaturases in plants.

Identification of Other ω3-desaturases

The Micromonas CCMP1545 filtered protein models genome sequence producedby the US Department of Energy Joint Genome Institute(http://www.jgi.doe.gov/) was analysed with the BLASTP program using SEQID NO:17 as the query sequence. This analysis revealed the presence of agene in Micromonas CCMP1545 (EuGene.0000150179) that had homology withSEQ ID NO:17. The open reading frame sequence is provided in SEQ IDNO:19 and the protein sequence is provided in SEQ ID NO:20.

BLAST analysis indicated that the full-length amino acid sequence SEQ IDNO:20 has homology with FAT-1, FAT-2 and ω3 desaturases. The maximumdegree of identity between the Micromonas CCMP1545 desaturase and otherproteins in the Genbank database (BLASTP) was 59% along the full lengthwith SEQ ID NO:17. This front-end desaturase contained a Δ12-FADS-likeconserved domain (NCBI conserved domain cd03507). Proteins containingboth of these domains are typically front-end desaturases required forthe synthesis of fatty acids, including the ω3 desaturase family. Wepredict that this protein will also function as an ω3 desaturase withΔ17-desaturase activity in planta.

Example 7 Isolation and Characterisation of Further Genes EncodingΔ9-elongase from Microalgae

Isolation and Characterisation of the Emiliania huxleyi CCMP1516Δ9-elongase

The Emiliania huxleyi CCMP1516 filtered protein models genome sequenceproduced by the US Department of Energy Joint Genome Institute(http://www.jgi.doe.gov/) was analysed with the BLASTP program using theamino acid sequence of GenBank Accession No. AF390174 as the querysequence. This analysis revealed the presence of a predicted gene inEmiliania huxleyi CCMP1516 that had homology with AF390174. The proteinsequence is provided in SEQ ID NO:28 and the encoding nucleotidesequence as SEQ ID NO:27. BLAST analysis indicated that the full-lengthamino acid sequence has homology with PUFA elongases. The maximum degreeof identity between the Emiliania huxleyi CCMP1516 elongase and otherproteins (BLASTP) was 80% with AF390174. The conserved GNS1/SUR4 familydomain (NCBI conserved domain pfam01151) was represented in thissequence, which typically indicated that the protein was involved inlong chain fatty acid elongation systems.

The Emiliania huxleyi CCMP1516 predicted protein sequence was used todesign and synthesize a codon-optimized nucleotide sequence that wasmost suitable for expression in dicotyledonous plants such as Brassicanapus (SEQ ID NO:29). The plasmid construct was designated0835668_Emihu-d9E_pMA.

Isolation and Characterisation of the P. pinguis and P. salinaΔ9-elongases

To identify possible conserved regions within the P. pinguis and P.salina Δ9-elongases an alignment was carried out of deduced elongaseamino acid sequences from the E. huxleyi Δ9-elongase, PLL00000665 (a P.lutheri EST sequence from TBcstDB identified by BLAST analysis using theE. huxleyi elongase sequences as query) and Genbank accession AAL37626(I. galbana Δ9-elongase). This revealed the consensus amino acidsequence blocks VDTRKGAYR (SEQ ID NO:76) and FIHTIMYTY (SEQ ID NO:77)corresponding to amino acid positions 40-48 and 170-178, respectively,of Emihu-d9E. The degenerate primers 5′-TGGTGGACACAAGGAAGGGNGCNTAYMG-3′(SEQ ID NO:78) and 5′-GTAGGTGTACATGATGGTRTGDATRAA-3′ (SEQ ID NO:79) weresynthesised based on the sequences of these two blocks and RT-PCR andPCR amplifications using RNA from P. pinguis and a cDNA library from P.salina (Zhou et al., 2007) was carried out using the Superscript III™Platinum® One-Step RT-PCR system or Taq DNA polymerase (NEB, Ipswich,Mass., USA).

A 641 basepair amplicon was generated from P. pinguis by RT-PCR, ligatedinto pGEM-T Easy® and sequenced. Primers were designed to extend the 641basepair fragment by 5′- and 3′-RACE, the 3′ end of the gene beingisolated by RT-PCR using the gene specific forward primer5′-GTCCTTGCTCCAGGGCTTCCACCA-3′ (SEQ ID NO:80) and the oligo-dT-SP6reverse primer 5′-ATTTAGGTGACACTATAGTTTTTTTTTTTTTTTTTT-3′ (SEQ IDNO:81). This product was diluted 1:10 and 1.0 μl used as template in asecond round of PCR using Taq DNA polymerase (NEB) with the genespecific forward primer 5′-TTCCAGAACGAGGGCATCTACGT-3′ (SEQ ID NO:82) andthe same reverse primer. A 1079 basepair amplicon was generated, ligatedinto pGEM-T® Easy and sequenced. The 5′ end of the gene was isolatedfrom 1.0 μg of P. pinguis cDNA generated with the gene specific reverseprimer 5′-TTGGGTGATCTGCATGAGCGTGATG-3′ (SEQ ID NO:83) and A-tailed byterminal transferase. This cDNA was then used as template for a PCRreaction using the oligo-dT-SP6 primer and the gene specific primer5′-CGAATACTTGAAGAGCTTGTTGGAGA-3′ (SEQ ID NO:84). This product wasdiluted 1:10 and 1.0 μl used as template in a second round of PCR usingthe oligo-dT-SP6 primer and the gene specific primer5′-GGGCTACGAGCTGGCAGATGAAGCA-3′ (SEQ ID NO:85). A 323 basepair ampliconwas generated, ligated into pGEM-T® Easy and sequenced. The full lengthsequence was assembled from the three partial sequences. The full lengthcoding region with a short region of 5′ UTR was amplified from total RNAby RT-PCR using the forward primer 5′-GAAAAAATGGTTGCGCCACCCATCA-3′ (SEQID NO:86) and the reverse primer 5′-TCACTACTTCTTCTTCTTGCCCGCGGC-3′ (SEQID NO:87). An 828 basepair amplicon, Pavpi-Elo1, was generated and thiswas ligated into pGEM-T® Easy and sequenced (SEQ ID NO:93). The deducedamino acid sequence of the P. pinguis Δ9-elongase is provided as SEQ IDNO:94.

Similarly, a 425 basepair amplicon was generated from P. salina by PCRusing the degenerate primers, ligated into pGEM-T Easy® and sequenced.Primers were designed to extend the 425 basepair fragment by 5′- and3′-RACE, the 3′ end of the gene being isolated by RT-PCR using the genespecific forward primer 5′-TTCCGGTACTCAGCGGTGGCG-3′ (SEQ ID NO:88) andthe oligo-dT-SP6 reverse primer. A 776 basepair amplicon was generated,ligated into pGEM-T® Easy and sequenced. The 5′ end of the gene wasisolated by PCR from the P. salina cDNA library using the M13R primer5′-CAGGAAACAGCTATGAC-3′ (SEQ ID NO:89) and a gene specific reverseprimer 5′-ACGTAGATGCCCTCGTTCTG-3′ (SEQ ID NO:90) with PfuUltra II®Fusion DNA polymerase as specified by the manufacturer. A 710 basepairamplicon was generated, ligated into pGEM-T® Easy and sequenced. Thefull length sequence was assembled from the three partial sequences. Thefull length coding region with a short region of 5′ UTR was amplifiedfrom total RNA by RT-PCR using the forward primer5′-CACCGAATGGCGACTGAAGGGATGCC-3′ (SEQ ID NO:91) and the reverse primer5′-CTACTCGGTTTTCATGCGGTTGCTGGA-3′ (SEQ ID NO:92). An 846 basepairamplicon, Pavsa-Elo3, was generated and this was ligated into pGEM-T®Easy and sequenced (SEQ ID NO:95). The deduced amino acid sequence ofthe P. salina Δ9-elongase is provided as SEQ ID NO:96.

Function Characterisation of the Δ9-elongases in Plant Cells

The entire coding regions of the Emiliania elongase (Emihu-d9E), Pavlovapinguis elongase (Pavpi-d9E) and Pavlova salina elongase (Pavsa-d9E),contained within EcoRI fragments, from plasmids 0835668_Emihu-d9E_pMA,pGEMT+Pavpi-d9E and pGEMT+Pavsa-d9E, respectively, were inserted into35S-pORE04 at the EcoRI site to generate 35S:Emihu-d9E (designatedpJP3027), 35S:Pavpi-d9E (designated pJP3103), 35S:Pavsa-d9E (designatedpJP3081) and 35S:Isoga-d9E (designated pJP2062). The enzyme activitiesof Emihu-d9E, Pavpi-d9E and Pavsa-d9E along with Isoga-d9E (Qi et al.,2002), used here as a positive control sample, were demonstrated inplanta using an enhanced Nicotiana benthamiana transient expressionsystem as described in Example 1.

These chimeric vectors were introduced into Agrobacterium tumefaciensstrain AGL1 and cells from cultures of these infiltrated into leaftissue of Nicotiana benthamiana plants in the greenhouse. The plantswere grown for a further five days after infiltration before leaf discswere taken for GC analysis which revealed, by presence of the productfatty acid, that both genes were functioning as Δ9-elongases in plantcells such as Nicotiana benthamiana.

Leaf tissue transformed with the Emiliania huxleyi CCMP1516 Δ9-elongasecontained 20:2^(Δ11,14) (6.6%) and 20:3^(Δ11,14,17) (6.4%), whichrepresented conversion efficiencies from LA and ALA of 39.9% and 12.4%,respectively. Leaf tissue transformed with the Pavlova pinguisΔ9-elongase contained 20:2^(Δ11,14) (10.1%) and 20:3^(Δ11,14,17) (6.6%),which represented conversion efficiencies of 56.0% and 13.3%,respectively. Leaf tissue transformed with the Pavlova salinaΔ9-elongase contained 20:2^(Δ11,14) (7.7%) and 20:3^(Δ11,14,17) (4.6%),which represented conversion efficiencies of 45.0% and 9.2%,respectively. Leaf tissue transformed with the Isochrysis galbanaΔ9-elongase contained 20:2^(Δ11,14) (9.2%) and 20:3^(Δ11,14,17) (7.5%),which represented conversion efficiencies of 48.9% and 15.4%,respectively (Table 9).

TABLE 9 Fatty acid composition (percent of total fatty acids) ofNicotiana benthamiana leaf tissue transiently transformed withΔ9-elongases. The standard deviations between separate infiltrationsperformed in triplicate are shown. Emihu- Pavsa- Pavpi- Isoga- Fattyacid Control Δ9E Δ9E Δ9E Δ9E Usual FA 16:0 15.7 ± 0.6 14.6 ± 0.1  15.2 ±0.6  14.5 ± 0.8  14.2 ± 0   16:1^(Δ3t) 1.5 ± 0  1.4 ± 0   1.3 ± 0.1 1.3± 0   1.3 ± 0.1 16:3^(Δ9,12,15)  6.8 ± 0.7 6.5 ± 0.8 6.1 ± 0.8 6.1 ± 1.47.4 ± 0.5 18:0  3.0 ± 0.1 3.4 ± 0.2 4.2 ± 0.2 3.4 ± 0.5 3.1 ± 0.218:1^(Δ9) 2.2 ± 0  2.9 ± 0.2 3.2 ± 0.2 3.6 ± 0.7 3.2 ± 0.1 18:2^(Δ9,12)11.8 ± 0.4 9.9 ± 0.1 9.4 ± 0.2 7.9 ± 0.5 9.7 ± 0.4 18:3^(Δ9,12,15) 56.0± 1.4 45.3 ± 2.2  46.0 ± 1.4  43.4 ± 1.8  41.5 ± 1.3  Other minor 3.0 ±0  3.0 ± 0   2.3 ± 0   3.1 ± 0   2.9 ± 0 Total 100  87.0 87.7 83.3 83.3New ω6 PUFA 20:2^(Δ8,11) — 6.6 ± 1.0 7.7 ± 0.7 10.1 ± 0.7  9.2 ± 0.820:3^(Δ8,11,14) — — — — — 20:4^(Δ5,8,11,14) — — — — — 22:4^(Δ7,10,13,16)— — — — — 22:5^(Δ4,7,10,13,16) — — — — — Total 0  6.6  7.7 10.1  9.2 Newω3 PUFA 20:3^(Δ11,14,17) — 6.4 ± 1.5 4.6 ± 0.7 6.6 ± 0.4 7.5 ± 0.220:4^(Δ8,11,14,17) — — — — — 20:5^(Δ5,8,11,14,17) — — — — —22:5^(Δ7,10,13,16,19) — — — — — 22:6^(Δ4,7,10,13,16,19) — — — — — Total0  6.4  4.6  6.6  7.5 Total new FA 0 13.0 12.3 16.7 16.7 Total FA 100 100   100   100   100  

The apparently high preference for the ω3 substrate ALA in the leaftissue was expected since the bulk of the substrate ALA in N.benthamiana leaf is located in the plastid and thus unavailable forextra-plastidial elongation and since both the plastidial andcytoplasmic ALA are isolated from the leaf during direct methylation theω3 conversion ratio artificially reduced. The E. huxleyi and I. galbanaΔ9-elongases displayed identical substrate preferences in N. benthamianawith ω3 to ω6 conversion ratios of 0.31. The most efficient conversionin the ω6 pool was seen with the P. salina Δ9-elongase with 56.0% ofsubstrate being converted. In contrast, 13.3% of the ω3 substrate wasconverted, a ratio of 0.24. The P. pinguis enzyme displayed the highestpreference for ω6 substrates with a conversion ratio of 0.20 resultingfrom 45.0% ω6 conversion but only 9.2% ω3 conversion.

Example 8 Construction of a Biosynthetic Pathway Including Δ9 Elongaseto Yield ARA

Construction of a Transgenic Delta-9 Elongase Pathway

A binary vector containing the Isochrysis galbana Δ9-elongase (aminoacid sequence GenBank Accession No. AF390174—open reading frame providedas SEQ ID NO:21, amino acid sequence as SEQ ID NO:22), Pavlova salinaΔ8-desaturase (Accession No. ABL96296—open reading frame provided as SEQID NO:23, amino acid sequence as SEQ ID NO:24) and Pavlova salinaΔ5-desaturase (Accession No. ABL96295—open reading frame provided as SEQID NO:25, amino acid sequence as SEQ ID NO:26) was constructed from thebinary vector pJP101acq. The design of this vector without the geneinserts is shown schematically in FIG. 14.

First, the SmaI-EcoRV fragment of a pBluescript clone containing theIsochrysis galbana Δ9-elongase was ligated into the SmaI site of pJP101acq to yield pJP105. The XhoI fragment of a pBluescript clone containingthe Pavlova salina Δ5-desaturase was ligated into the XhoI site ofpJP105 to yield pJP106. The NotI fragment of a pBluescript clonecontaining the Pavlova salina Δ8-desaturase was ligated into the NotIsite of pJP106 to yield pJP107, which is shown schematically in FIG. 15.

Several points are notable about the design. Firstly, two of the threegenes were transcribed divergently on the T-DNA, i.e. away from eachother. This was done to prevent transcription from either gene beingdirected toward, and potentially interfering with, expression of theother gene and thereby maximising expression of both. Secondly, thethird gene in the genetic construct, in this case encoding theΔ8-desaturase, was spaced apart from the second gene oriented in thesame direction, encoding the Δ9-elongase, by the insertion of a spacer.It was thought that a distance of at least 1.0 kb between the stop codonof the upstream gene and the start codon of the downstream gene wouldreduce the risk of transcription of the former interfering with thelatter, or potentially causing gene silencing. Thirdly, the 5′-UTR ofeach of the three genes was modified to include a TMV leader sequencewhich was known to provide for efficient translation. Any other 5′UTRsequence which is known to confer high translation efficiency could havebeen used instead of the TMV sequence.

pJP107 was introduced into Agrobacterium strain AGLI by electroporationand the transformed strain used to introduce the genetic construct intoArabidopsis thaliana, ecotype MC49, which was a fad3/faeI mutant withhigh levels of LA as potential beginning fatty acid substrate for theΔ8-desaturase. Plant transformation and analysis was carried out usingthe floral dipping method (Clough and Bent, 1998). Seeds (T1 seeds) fromthe treated plants (T0 plants) were plated out on hygromycin (20 mg/L)selective media and transformed plants were selected and transferred tosoil to establish 24 confirmed T1 transgenic plants. Most of these T1plants were expected to be heterozygous for the introduced geneticconstruct. T2 seed from the 24 transgenic plants were collected atmaturity and analysed for fatty acid composition. These T2 linesincluded lines that were homozygous for the genetic construct as well asones which were heterozygous. T2 plants were established from the T2seed for the 6 lines containing the highest ARA levels, using selectionon MS medium containing hygromycin (20 mg/mL) to determine the presenceof the transgenes. For example, the T2 seeds were planted from the T1plant designated FW-10, containing 5.8% ARA and showing a 3:1segregation ratio of resistant to susceptible progeny on the hygromycinmedium, indicating that FW-10 contained the genetic construct at asingle genetic locus. The fatty acid profiles of T3 seed lots from FW-10were analysed and the data are presented in Table 10.

TABLE 10 Fatty acid composition of Arabidopsis seed transformed with thegenetic construct pJP107 containing the Isochrysis galbana Δ9-elongase,Pavlova salina Δ8-desaturase and Pavlova salina Δ5-desaturase genes.Control FW10-23 MC49 Sample P1235 P1254 14:0 0.0 0.1 16:1ω7 0.6 0.4 16:09.5 8.4 18:2 ω6 30.9 50.9 18:3 ω3 0.0 1.0 18:1 ω9 21.4 30.9 18:1 ω7 3.33.5 18:0 4.3 3.4 20:4 ω6 21.0 0.0 20:5 ω3 1.3 0.0 20:3 ω6 1.1 0.0 20:4ω3* 0.2 0.0 20:2 ω6 2.6 0.0 20:3 ω3 0.2 0.0 20:1 ω9/ω1 1.6 0.2 20:0 0.70.5 22:4 ω6 0.8 0.0 22:5 ω3 0.0 0.0 22:0 0.0 0.2 24:1 ω11/13 0.2 0.124:0 0.2 0.2 Sum 100 100 Sum ω6 PUFA 56 51 % conversions Δ9E LA → 20:2ω6 45 0 ALA → 20:3 ω3 100 0 Δ8D 20:2 ω6 → 20:3 ω6 90 0 20:3 ω3 → 20:4 ω388 0 Δ5D 20:3 ω6 → 20:4 ω6 95 0 20:4 ω3 → 20:5 ω3 90 0

As summarised in Table 10, seed of untransformed Arabidopsis (ecotypeMC49) contained significant amounts of the precursor ω6 substrate LA butdid not contain any ARA or the intermediate fatty acids expected tooccur along the Δ9 elongase pathway. In contrast, seed from transformedplant FW 10-23 containing the pJP107 construct contained significantlevels of 20:2n-6, 20:3n-6 and 20:4n-6 (ARA), including 21% ARA, theproduct of the three enzymatic steps starting with LA. Furthermore, thelow level of ALA in the seedoil (1.0% in control MC49) was veryefficiently converted to EPA, which was present at a level of 1.3% intransformed line FW10-23.

Discussion: Conversion Efficiencies and Biochemical Implications

The relative efficiencies of the individual enzymatic steps encoded bythe pJP107 construct could be assessed by examining the percentageconversion of substrate fatty acid to product fatty acids (includingsubsequent derivatives) in FW-10-23. In the ω6 pool, the Isochrysisgalbana Δ9 elongase showed 45% conversion of LA to EDA and subsequentlydesaturated fatty acids. In the same seed, the Pavlova salinaΔ8-desaturase and Δ5-desaturase showed conversion efficiencies of 90%and 95%, respectively of the ω6 fatty acids to their relevant products.In comparison, in the ω3 pool, the Isochrysis galbana Δ9 elongase showedessentially 100% conversion of ALA to elongated products, whilst thePavlova salina Δ8-desaturase and Δ5-desaturase showed conversionefficiencies of 88% and 90%, respectively. These enzymatic stepsresulted in the synthesis of 1.3% EPA, even though the Arabidopsisthaliana MC49 background contains only low levels of ALA. In the mostdramatic result, it was noted that ALA was not detected in the seedoil,indicating essentially 100% conversion of ALA to elongated products ofALA by the Δ9 elongase.

It is interesting to note that the levels of unusual intermediate fattyacids found in FW-10-23 were low (<0.4% in the ω3 pool) and comparableto those already found in the food-chain in various seafoods (Table 11).Even though untransformed MC49 seedoil contained only low levels of ALAand this might have contributed to the low observed levels of, forexample, the intermediate fatty acid ETrA, it is predicted that when thesame pathway is assembled in a genetic background having higher ALAlevels, the resultant seedoil would still have relatively low levels(<3%) of ETrA. The presence of such low levels of these intermediateswas likely due to the very efficient desaturation of the Δ9 elongatedintermediates.

TABLE 11 Comparison of the fatty acids in Arabidopsis seed transformedwith the genetic construct pJP107 containing the Isochrysis galbanaΔ9-elongase, Pavlova salina Δ8-desaturase and Pavlova salinaΔ5-desaturase genes and the intermediate fatty acids found in a range ofseafood samples. Seafood range- Sample P1235 Mean maximum 14:0 0.0 1.731.1 16:1 ω7 0.6 2.9 8.2 16:0 9.5 18.7 53.6 18:2 ω6 30.9 1.9 14.6 18:3ω3* 0.0 0 0 18:1 ω9 21.4 13.9 59.5 18:1 ω7 3.3 3 7.9 18:0 4.3 8.5 14.720:4 ω6 21.0 6.7 19.1 20:5 ω3 1.3 7.1 22.2 20:3 ω6 1.1 0.3 1.5 20:4 ω30.2 0.5 2.8 20:2 ω6 2.6 0.4 1.8 20:3 ω3* 0.2 0 0 20:1 ω9/ω11 1.6 2.221.1 20:0 0.7 0.4 4.2 22:4 ω6 0.8 1 4.4 22:5 ω3 0.0 2.4 14.9 22:0 0.00.2 0.7 24:1 ω{tilde over (9)}11/13* 0.2 0 0 24:0 0.2 0.2 1.6 Sum ω6PUFA 56 10 41 % conversions Δ9E LA-->20:2 ω6 45 82 ALA-->20:3 ω3 100 100Δ8D 20:2w6-->20:3 ω6 90 95 20:3w3-->20:4 ω3 88 100 Δ5D 20:3w6-->20:4 ω695 96 20:4w3-->20:5 ω3 90 95

It is worth noting that the Pavlova salina Δ8-desaturase wasconsiderably more efficient in converting ETrA to ETA than otherreported Δ8-desaturases, in particular when co-expressed with the Δ9elongase and Δ5 desaturase. For example, it has been reported that whenthe Euglena gracilis Δ8-desaturase was co-expressed with either theEuglena gracilis or the Isochrysis galbana Δ9-elongase in soybeanembryos, the conversion efficiencies of ω3 and ω6 substrates were 64%and 73%, respectively. The efficiency of each step observed in theexperiment described above and the overall conversion efficiency of ALAto EPA was also much higher than that reported by Qi et al. (2004) inArabidopsis leaves, where they observed only 3.0% EPA and substantiallevels of the undesirable intermediates including ETrA (4.6%).

Elongases are known to only access substrates in the acyl-CoA pool. Thefact that the subsequent Δ8-desaturase and Δ5-desaturase steps wereobserved to function at extremely high efficiency in the transformedseeds even though the Δ9-elongated product was undoubtedly produced inthe acyl-CoA pool was a strong indication that both of the Pavlovasalina desaturases were able to access acyl-CoA substrates with highefficiencies.

Biosynthesis of High ARA and EPA Levels Using the 49-elongase Pathway

From these data and the observations on efficiency of the individualsteps, it was predicted that it would be possible to generate highlevels of ARA and EPA and subsequently DPA and DHA in a transgenicplants such as Arabidopsis, canola, soybean, linseed or cotton using amodified Δ9-elongase pathway. It was further predicted that even higherlevels can be made with further addition of any one of three enzymaticfunctions, namely an acyl-CoA Δ12-desaturase function to increase theamount of available substrate LA in the acyl-CoA pool for Δ9-elongation,secondly the addition of a Δ15-desaturase to increase the level of ALAfor direct conversion to EPA, and thirdly a Δ17-desaturase which canconvert ARA to EPA such as the one described in Example 6. Morepreferably, the addition of both an acyl-CoA Δ12-desaturase and eitherthe Δ15-desaturase or the Δ17-desaturase would provide maximal levels.Thus, use of enzymes capable of accessing substrates in the acyl-CoApool is expected to result in more efficient conversion to EPA, DHA andDHA.

The observed synthesis of 1.3% EPA was remarkable and unexpectedconsidering that the Arabidopsis thaliana MC49 background contained afad3 mutation which resulted in low levels of ALA accumulation (1-3%).We predict that when this, or similar, Δ9-elongase pathways(Δ9-elongase, Δ8-desaturase and Δ5-desaturase) are transformed into aplant containing high levels of ALA, high levels of EPA will result. Forexample, we predict that transformation of this pathway into anArabidopsis line overexpressing the Perilla frutescens Δ15-desaturase orother Δ15-desaturase genes will result in EPA levels of at least 25% ofthe total fatty acid in seedoil.

Example 9 Expression of PUFA Pathway Genes in Plant Cells

One alternative to the stable transformation of plants is the transientexpression of transgenes in leaves, such as that first introduced byKapila et al (1997). With this technique, nuclei of permissive leafcells (Zipfel et al., 2006) are transformed via infiltration of abaxialair-spaces with Agrobacterium cultures harbouring expression constructswithin T_(DNA) borders. Expression of transgenes in leaves issignificantly enhanced by the co-introduction of viral suppressorproteins, such as P19 (Voinnet et al., 2003) and HC-Pro (Johansen andCarrington, 2001; Kasschau et al., 2003), that inhibit the host cells'transgene silencing apparatus and extend transgene expression over alonger period of time.

Leaves have a complex lipid metabolism that is dominated by the largepools of plastidial galactolipids monogalactosyl diacylglycerols (MGDG)and digalactosyl diacylglycerols (DGDG). More minor pools of fatty acidsexist outside the plastidial compartments including those esterified tophospatidylcholine (PC), coenzyme A (CoA) and mono- anddi-acylglycerides (MAG, DAG; Ohlrogge and Browse, 1995). The enzymes ofLC-PUFA synthesis used in this Example reside on the endoplasmicreticulum (ER; Napier, 2007) where they have access to the relativelyminor leaf lipid pools esterified to PC and CoA. Metabolic products ofPC-CoA-linked reactions, such as those active on the ER, can beaccumulated in triacylglycerides (TAG) by the overexpression of adiacylglyceride-O-acyltransferase (DGAT; Bouvier-Nave et al., 2000).Compared to MAG or DAG, fatty acids residing on TAG are moremetabolically inert and are less likely to re-enter lipid biosynthesispathways or traffic into plastids. Importantly, TAG can be readilyseparated from the more abundant lipid classes residing in leaf plastidsusing standard thin-layer chromatography (TLC) techniques. Therefore acombination of enhanced TAG accumulation and TAG/lipid classpurification could be helpful to more fully understand the LC-PUFAenzyme reactions on the leaf ER.

This system was tested for production of LC-PUFA using genes encodingdesaturases and elongases in this Example.

Plasmid Constructs for Transient Expression

Binary vectors were prepared by cloning the coding region of the geneinto a modified version of the pORE04 binary vector described by Coutuet al (2007) in which the Cauliflower Mosaic Virus (CaMV) 35S promoterhad been cloned into the SfoI site to yield 35S-pORE04. The I. galbanaΔ9-elongase gene coding region (Genbank accession AAL37626) (SEQ IDNO:21) was amplified from genomic DNA cloned into 35S-pORE04 at theEcoRI site. Plant expression codon-optimised versions of the three P.salina desaturases (Genbank accessions ABL96296, ABL96295 andAAY15136—each described in WO 2005/103253) were cloned into theEcoRV-SmaI sites of 35S-pORE04 as SwaI inserts. The non-optimised P.salina Δ5-elongase (Genbank accession AAY15135) was cloned as anXhoI-XbaI fragment into 35S-pORE04 at the XhoI-NheI sites. A CaMV35S-driven version of the P19 viral suppressor was kindly donated by DrPeter Waterhouse. The A. thaliana DGAT1 gene coding region (Genbankaccession AAF19262) (SEQ ID NO:74) was obtained by RT-PCR and was clonedas a BamHI-EcoRV fragment into the corresponding sites in 35S-pORE04.Total RNA was isolated from Phytophthora infestans using a RNeasy minikit (QIAGEN) and Platinum Superscript III One-Step (QIAGEN) RT-PCRperformed. The resulting amplicon which contained the P. infestansΔ17-desaturase protein coding region (WO 2005/012316) was cloned intopGEMT-Easy (Promega) and sequenced. The EcoRI fragment was then clonedinto 35S-pORE04.

Agrobacteria infiltrations and N. benthamiana Growth Conditions

Agrobacterium tumefaciens strain AGL1 harbouring each binary vector wasgrown at 28° C. in LB broth supplemented with the appropriateantibiotics. Cultures were centrifuged and gently resuspended in twovolumes of infiltration buffer (5 mM MES, 5 mM MgSO₄, pH 5.7, 100 μMacetosyringone) and grown for a further 3 hours. Optical densities ofeach culture were measured and a final combination of cultures preparedso that each Agrobacterium construct equaled OD_(600nm) 0.2 or asotherwise indicated for FIG. 16. The cells were infiltrated, asdescribed by Voinnet et al. (2003), into the underside of leaves of onemonth-old N. benthamiana plants that had been housed in a 23° C. plantgrowth room with 10:14 light:dark cycle. Infiltrated areas were circledby a permanent marker. Following infiltration, the plants were left at28° C. for an hour after which they were transferred to a 24° C. plantgrowth room until analysis. Unless otherwise indicated, all N.benthamiana agroinfiltrations were performed in the presence of aseparate binary construct containing the P19 viral suppressor protein.

Lipid Analysis

The fatty acid profiles of leaf tissues, or lipid class samples wereanalysed in this Example by GC and GC-MS after transmethylating using asolution of methanol/HCl/dichloromethane (DCM; 10/1/1 by volume) at 80°C. for 2 hr to produce fatty acid methylesters (FAME). The FAME wereextracted in hexane:DCM (4:1, v/v) and reconstituted in DCM prior toanalysis by GC and GC-MS.

For lipid class analysis, total lipids were extracted two times fromapproximately 50 mg fresh weight of infiltrated leaf tissue using themethod described by Bligh and Dyer (1959). Neutral lipids were purifiedby TLC on precoated silica gel plates (Silica gel 60, Merck) withhexane/diethyl ether/acetic acid (70/30/1 by vol.), while polar lipidswere fractionated using two-dimensional TLC, chloroform/methanol/water(65/25/4 by vol.) for the first direction andchloroform/methanol/NH₄OH/ethylpropylamine (130/70/10/1 by vol.) for thesecond direction (Khozin et al., 1997). The lipid spots were visualizedby iodine vapour, collected into vials and transmethylated to produceFAME for GC analysis as described above. TAG was quantified as the totalamount of fatty acids present, which was estimated by GC analysis asmentioned above and according to known amount of external standardsinjected for each fatty acid.

GC was performed using an Agilent Technologies 6890N GC (Palo Alto,Calif., USA) equipped with a non-polar Equity™-1 fused silica capillarycolumn (15 m×0.1 mm i.d., 0.1 μm film thickness), an FID, asplit/splitless injector and an Agilent Technologies 7683 Seriesautosampler and injector using helium as the carrier gas. Samples wereinjected in splitless mode at an oven temperature of 120° C. and afterinjection the oven temperature was raised to 201° C. at 10° C.min⁻¹ andfinally to 270° C. and held for 20 min. Peaks were quantified withAgilent Technologies ChemStation software (Rev B.03.01 (317), Palo Alto,Calif., USA). Peak responses were similar for the fatty acids ofauthentic Nu-Check GLC standard-411 (Nu-Check Prep Inc, MN, USA) whichcontains equal proportions of 31 different fatty acid methyl esters,ranging from octanoate to DHA and several other LC-PUFAs. Slightvariations of peak responses among peaks were balanced by multiplyingthe peak areas by normalization factors of each peak. The proportion ofeach fatty acid in total fatty acids was calculated on the basis ofindividual and total peaks areas of the fatty acids.

GC-MS was performed to confirm the identity of all new fatty acidsformed and was carried out on a Finnigan GCQ Plus GC-MS ion-trap fittedwith on-column injection set at 45° C. Samples were injected using anAS2000 autosampler onto a retention gap attached to a non-polar HP-5Ultra 2 bonded-phase column (50 m×0.32 mm i.d.×0.17 μm film thickness).The initial temperature of 45° C. was held for 1 minute, followed bytemperature programming an increase of 30° C.min⁻¹ to 140° C. then at 3°C.min⁻¹ to 310° C. where it was held for 12 minutes. Helium was used asthe carrier gas. Mass spectrometer operating conditions were: electronimpact energy 70 eV; emission current 250 μamp, transfer line 310° C.;source temperature 240° C.; scan rate 0.8 scans.s⁻¹ and mass range40-650 Dalton. Mass spectra were acquired and processed with Xcalibur™software.

Modifying the N. benthamiana Fatty Acid Profile with aTransiently-expressed Fatty Acid Elongase

To estimate the concentration of Agrobacterium required to generatemaximal production of a functional transgenic enzyme, a gene wasexpressed which encodes the Isochrysis galbana Δ9-elongase (IgΔ9elo; Qiet al., 2002), which was known to act on the CoA-linked linoleic acid(LA) and ALA substrates known to be abundant in N. benthamiana leaves.Following transfer of this gene into a binary vector downstream of theCauliflower Mosaic Virus (CaMV) 35S promoter, this construct wasagroinfiltrated into N. benthamiana leaves in the presence of the P19viral suppressor protein to suppress host-mediated transgene silencingand the level of Δ9-elongation assessed. The elongation products of LAand ALA, EDA and ETrA, respectively, were detected with near maximalgene activity obtained with Agrobacterium cultures having OD₆₀₀=0.2(FIG. 16). It was interesting to note, however, that agroinfiltrationsof quite dilute concentrations of the culture (as low as OD₆₀₀=0.05)also resulted in readily detectable levels of enzyme activity.

Effect of Transient DGAT Expression on Triacylglycerol Accumulation

It was next investigated whether the size of the TAG pool in N.benthamiana leaves could be increased to provide a larger sink in whichto capture the products of the introduced fatty acid biosyntheticenzymes acting on the ER. A construct containing the Arabidopsisthaliana DGAT1 (AtDGAT1) gene which catalyses the last step in TAGbiosynthesis by the Kennedy pathway was tested as a possible means toincrease the TAG pool in leaves, since leaves naturally produce only lowlevels of TAGs. The construct was introduced into N. benthamiana leavesby agroinfiltration as above. To test for the presence of TAGs, segmentsof infiltrated leaves approximately 1 cm² in size were submerged in asmall Petri dish containing 1% aqueous Nile Blue (BDH, Poole, UK),vacuum-infiltrated for 3 minutes, rinsed briefly in water, incubated in1% acetic acid for 3 minutes, and mounted in water for observation.Fluorescence emission was collected at 570-670 nm using a 488 nmexcitation on a Leica SP2 laser scanning confocal microscope (LeicaMicrosystems, Sydney, Australia). Untransformed sectors of the sameleaves were used as controls. The relative amounts of TAG accumulationin each assay were estimated using ImageJ software.

Transient expression of the Arabidopsis thaliana DGAT1 (AtDGAT1)resulted in the production of significantly more lipid bodies that werestained with Nile Blue and observed using confocal microscopy. Theincrease in TAG was quantified by fractionating the total lipids intoTAG, using neutral phase TLC separation from other leaf lipid classes,after which the amount of TAG was measured as the amount of total fattyacids in the TAG fraction. Transient expression of P19 or P19 andAtDGAT1 together resulted in an increase in TAG from 46 μg·g⁻¹ freshweight to 206 μg·g⁻¹ fresh weight, respectively, showing that additionof the DGAT1 gene increased the levels of TAG that accumulated in leaftissue. Therefore, this gene was included in subsequent experimentsunless indicated otherwise.

Availability of Exogenous Fatty Acid Substrates to Transiently ExpressedGenes

It was next examined whether an exogenous fatty acid substrate that wasnot native to N. benthamiana could be supplied to the leaf and becomeavailable for transgene-mediated conversion, thus allowing individualenzymatic steps to be tested in isolation. To test this, the geneencoding Phytophthora infestans Δ17-desaturase (PiΔ17des) which acts onARA, a substrate not naturally present in N. benthamiana, wasagroinfiltrated to produce EPA. Four days after infiltration withPiΔ17des, the leaf was fed an ARA-ammonium salt by injection in a mannersimilar to that performed to transform the leaf with Agrobacteriumcultures. The leaf was then allowed to metabolise the substrate for fourhours before the total lipids were extracted from the leaf tissue. GCand GC-MS analysis of these total lipids showed that 37% of theexogenously fed ARA was converted by Δ17-desaturation to EPA, anefficiency comparable to that reported in yeast-based assays (WO2005/012316).

Rapid Assembly of Five-step LC-PUFA Pathways from Separate BinaryVectors

Having established that the N. benthamiana system was a useful tool indetermining the function of a single transgene and enhanced TAGaccumulation, the extent to which the system could be used to assembleentire LC-PUFA pathways was investigated. In this study, genes encodingfive LC-PUFA metabolic enzymes were tested, that produce two parallellinear LC-PUFA pathways, namely the ω6-pathway, converting LA toDPA^(ω6), and the ω3-pathway, converting ALA to DHA (FIG. 1). Thebiosynthetic genes used were the Isochrysis galbana Δ9-elongase(IgΔ9elo), Pavlova salina Δ8-desaturase (PsΔ8des), P. salinaΔ5-desaturase (PsΔ5des), P. salina Δ5-elongase (PsΔ5elo) and P. salinaΔ4-desaturase (PsΔ4des; Qi et al., 2004; Robert et al., 2009; Zhou etal., 2007). Each gene was cloned separately into a plant binaryexpression vector downstream of the CaMV 35S promoter as described aboveand a mixture of these constructs, each present at a concentration ofOD_(600nm)=0.2, was agroinfiltrated into the abaxial surfaces of N.benthamiana leaves alongside AtDGAT1 and P19, making a total of sevenindividual constructs with a total OD_(600 nm)=1.4.

Five days after infiltration leaf discs were sampled and fatty acidmethyl esters (FAME) were produced directly from the fresh tissue andanalysed and identified by GC/MS (Table 12). It was clear that the allof the pathway enzymes were able to accept either the ω6 or ω3 PUFA assubstrates and that their sequential action on LA or ALA, led to thesynthesis of the LC-PUFA, ARA and DHA, respectively. A total percentageof newly-produced LC-PUFA of 16.9% was identified, including 9.8% ω6LC-PUFA and 7.1% ω6 LC-PUFA. Of all of these newly-formed LC-PUFA, ARA,EPA and DHA are considered nutritionally important and constituted 3.6%,2.6% and 1.1%, respectively, of the total fatty acids in the leaftissues. Enzymatic conversion efficiencies were calculated for each stepof the ω6 and ω3 pathways and compared to those from previous reports(FIG. 17). The first three steps of both the ω6 and ω3 five-steppathways were similar in efficiency compared to those described by Qi etal. (Qi et al., 2004), whilst the efficiencies of the last two steps ofthe pathways were the same as those used by Robert et al. (Robert etal., 2005). This comparison of transiently expressed genes and stablyexpressed genes indicated that both methods of introducing the pathwaysgenerate similar metabolic fluxes or efficiencies. These conversionefficiencies calculated on total fatty acid profiles are likely to be anunderestimate, especially for the first step, a Δ9-elongation, due tothe diluting effect of large LA and ALA pools in the plastid. This issuewas addressed by fractionation of lipid classes as follows.

TABLE 12 Fatty acid profiles of N. benthamiana leaf spots producing bothω6- and ω3-LC-PUFA. Each infiltration contained a mixture ofAgrobacterium cultures harbouring ectopic expression constructs of theP19 viral suppressor isolated from Tomato Bushy Stunt Virus and theArabidopsis thaliana diacylglycerol O-acyltransferase (AtDGAT1). LC-PUFApathway infiltrations include an extra five genes, namely, Isochrysisgalbana Δ9 elongase (IgΔ9elo), Pavlova salina Δ8-desaturase (PsΔ8des),P. salina Δ5-desaturase (PsΔ5des), P. salina Δ5-elongase (PsΔ5elo) andP. salina Δ4-desaturase (PsΔ4des). For clarity, saturated and minorfatty acids were not included in the table, but were used forcalculation of percentages. (−) indicates no detectable amounts of fattyacid. Data are generated from 3 replicates and standard errors areshown. Total FAME (%) 5 LC-PUFA Fatty acid Control genes 16:0 15.9 ±0.2  20.1 ± 0.9  16:1^(Δ3t) 1.7 ± 0.1 1.5 ± 0.2 16:3^(Δ9,12,15) 6.3 ±0.3 5.2 ± 0.3 18:0 3.6 ± 0.3 3.7 ± 0.2 18:1^(Δ9) 2.8 ± 0.1 3.1 ± 0.718:2^(Δ9,12) (LA) 18.6 ± 0.1  8.0 ± 0.7 18:3^(Δ9,12,15) (ALA) 45.5 ±1.4  38.0 ± 1.9  20:0 1.3 ± 0.4 0.8 ± 0   Other minor 4.1 2.6 Total 10083.1 New ω6 PUFA 20:2^(Δ11,14) (EDA) 0 1.4 ± 0.2 20:3^(Δ8,11,14) (DGLA)0 0.3 ± 0   20:4^(Δ5,8,11,14) (AA) 0 3.6 ± 0.4 22:4^(Δ7,10,13,16) (DTA)0 1.5 ± 0.1 22:5^(Δ4,7,10,13,16) (DPA^(ω6)) 0 3.0 ± 0.4 Total 0 9.8 Newω3 PUFA 20:3^(Δ11,14,17) (ETrA) 0 2.3 ± 0.1 20:4^(Δ8,11,14,17) (ETA) 00.2 ± 0   20:5^(Δ5,8,11,14,17) (EPA) 0 2.6 ± 0.3 22:5^(Δ7,10,13,16,19)(DPA^(ω3)) 0 0.9 ± 0.1 22:6^(Δ4,7,10,13,16,19) (DHA) 0 1.1 ± 0.1 Total 07.1 Total new fatty acids 0 16.9 Total fatty acids 100 100Lipid Class Partitioning of LC-PUFA

In order to assess the partitioning of the newly synthesised LC-PUFAbetween TAG phospholipids and plastidial galactolipids the total lipidsof N. benthamiana leaves transiently expressing the LC-PUFA pathwaygenes were subjected to lipid class fractionation as described above andtheir fatty acid profiles determined (Table 13). N. benthamiana leaflipids contain lipid classes and fatty acid profiles typical of leavesfrom higher plants (Fraser et al., 2004; Moreau et al., 1998). Bothnewly-synthesised ω6 and ω3 LC-PUFA were predominantly confined to lipidclasses typically found outside the plastid while the plastidial lipidswere essentially devoid of these fatty acids. For example, TAG andphospholipids (PC, PE and PA)—the dominant extraplastidial leaflipids—contained up to 20.4% and 16.9% of newly-synthesised ω6 and ω3LC-PUFA, respectively. Remarkably, leaves expressing the full LC-PUFApathways, AtDGAT1 and P19 produced TAG enriched with 37% of LC-PUFA. Ofparticular interest was the accumulation of the nutritionally importantfatty acids ARA, EPA and DHA, present at 7.2%, 5.9% and 3%,respectively, in leaf TAG. Fractionation revealed that the dominantplastidial lipid classes, MGDG, DGDG and PG, contained only 1.1% and0.3% of the newly synthesised ω6 and ω3 LC-PUFA, respectively. Althoughthese plastidial lipid classes represent the largest pools of fattyacids in the leaf, collectively, these classes contained only a smallamount of ω6 and ω3 LC-PUFA compared to TAG. Interestingly, the SQDGlipid class was totally devoid of the newly-synthesised LC-PUFA.

Lipid class fractionations were also used to calculate enzymaticefficiencies at each step of the LC-PUFA pathways that are associatedwith the ER for the fatty acids in TAG, which have no access to theplastidial lipids (FIG. 17). The removal of the plastidial lipid classesfrom these calculations had the most dramatic effect on theΔ9-elongation step for ALA into ETrA, increasing the conversionefficiencies from 16% to 55%. This three-fold increase in the enzymeconversion efficiency at this step is due to the large pools of ALA inplastids that are unavailable to this ER-bound enzyme (Table 13).

TABLE 13 Fatty acid profiles of N. benthamiana lipid classes expressingP19, AtDGAT1 and the five LC-PUFA genes as described in Table 12. Datafor TAG were generated from lipid class separations using 1-dimensionalTLC and the other lipid classes were separated on 2-dimensional TLC.Experiments were conducted in triplicate and standard errors are shown.Extra-Plastidial Plastidial Fatty acid TAG PC PE PA MGDG DGDG SQDG PG16:0 22.6 ± 0.6  24.3 ± 0.8  23.0 ± 0.4  22.5 ± 2.8  4.7 ± 0.5 20.9 ±0.7  52.7 ± 2.1  31.9 ± 1.9  16:1^(Δ3t) 0.2 ± 0   0 0 0.8 ± 0.7 0 0 0.5± 0.5 21.6 ± 1.5  16:3^(Δ9,12,15) 0.4 ± 0   0.1 ± 0.1 0 0 17.1 ± 1.0 1.4 ± 0.2 1.2 ± 0.1 0 18:0 7.8 ± 0.7 8.9 ± 1.5 8.6 ± 1.6 8.7 ± 2.2 0.8 ±0.1 3.8 ± 0.3 7.2 ± 1.1 5.2 ± 0.7 18:1^(Δ9) 3.2 ± 0.9 6.7 ± 2.3 1.8 ±0.6 5.6 ± 2.2 0.9 ± 0.1 1.3 ± 0.1 2.3 ± 0.3 11.8 ± 0.2  18:2^(Δ9,12)(LA) 9.2 ± 0.2 16.6 ± 1.2  21.2 ± 1.5  18.1 ± 0.6  2.7 ± 0.1 3.9 ± 0.15.3 ± 0.1 12.9 ± 0.5  18:3^(Δ9,12,15) (ALA) 13.8 ± 1.6  12.7 ± 1.6  13.6± 1.7  14.4 ± 1.2  70.5 ± 1.9  66.8 ± 0.9  30.7 ± 2.2  15.1 ± 1.8  20:02.2 ± 0.1 0.6 ± 0   1.0 ± 0.1 0.4 ± 0.3 0 0 0 0 Other minor 3.3 2.8 4.21.1 1.3 1.0 0.1 0.9 Total 62.7 72.7 73.4 71.6 98 99.1 100 99.4 New ω6PUFA 20:2^(Δ11,14) (EDA) 4.2 ± 0.3 3.2 ± 0.2 1.9 ± 0.1 2.8 ± 0.1 0.2 ±0   0.1 ± 0.1 0 0 20:3^(Δ8,11,14) (DGLA) 0.6 ± 0   0.8 ± 0   0.7 ± 0.10.5 ± 0.4 0 0 0 0 20:3^(Δ5,8,11,14) (AA) 7.2 ± 0.7 5.3 ± 0.3 8.4 ± 0.37.3 ± 0.2 0.5 ± 0.1 0 0 0.5 ± 0   22:4^(Δ7,10,13,16) (DTA) 1.7 ± 0.5 5.8± 0.1 3.7 ± 0.3 5.6 ± 0.2 0.2 ± 0   0 0 0.1 ± 0.2 22:5^(Δ4,7,10,13,16)6.7 ± 0.4 1.9 ± 0.1 2.5 ± 0.3 1.8 ± 0.4 0.2 ± 0   0.2 ± 0   0 0(DPA^(ω6)) Total new ω6 20.4 17.0 17.2 18.0 1.1 0.3 0 0.6 LC-PUFA New ω3PUFA 20:3^(Δ11,14,17) (ETrA) 6.7 ± 0.6 3.3 ± 0.2 2.2 ± 0.1 3.0 ± 0.2 0.5± 0.1 0.6 ± 0   0 0 20:4^(Δ8,11,14,17) (ETA) 0.5 ± 0   0.1 ± 0.2 0.1 ±0.2 0.1 ± 0.2 0 0 0 0 20:5^(Δ5,8,11,14,17) 5.9 ± 0.4 2.3 ± 0.3 3.4 ± 0.23.1 ± 0.3 0.3 ± 0   0 0 0 (EPA) 22:5^(Δ7,10,13,16,19) 0.8 ± 0   3.2 ±0.3 2.1 ± 0.1 3.2 ± 0.6 0.1 ± 0   0 0 0 (DPA^(ω3))22:6^(Δ4,7,10,13,16,19) 3.0 ± 0.2 1.4 ± 0.1 1.6 ± 0.2 1.0 ± 0.8 0 0 0 0(DHA) Total new ω3 16.9 10.3 9.4 10.4 0.9 0.6 0 0 LC-PUFA Total new 37.327.3 26.6 28.4 2.0 0.9 0 0.6 Total fatty acids 100 100 100 100 100 100100 100Discussion

These experiments showed that transient expression of a series ofpathway genes in N. benthamiana or other plant leaves mimickedexpression in stably-transformed plants and was therefore well-suitedand predictive of expression of the pathways in seeds for production ofLC-PUFA oils. The transient expression system provided aninterchangeable expression platform that gave rapid and reliable resultsfor the entire pathway, where single components could easily beexchanged in multi-step recombinant pathways. The transient assembly ofLC-PUFA biosynthesis was robust and reproducible. Assays were conductedin triplicate and produced tight data points, typically with a standarderror less than 5%.

The first step in the assembled LC-PUFA pathways, a Δ9 elongation,showed a higher rate of elongation of LA than ALA, as observedpreviously in stably-transformed plants (Fraser et al., 2004). Thisdifference cannot be accounted for by fatty acid substrate preferencesof the enzyme as expression in yeast showed the IgΔ9 elongase to haveequal preference for LA and ALA (Qi et al., 2002). It is more likelythat the higher rate of elongation observed for LA was a reflection ofthe existence of a higher amount of LA than ALA in the leafextraplastidial acyl-CoA pool—the site of action of the elongase(Domergue et al., 2003; Fraser et al., 2004).

Lipid class analysis demonstrated that all of the newly-formed LC-PUFAwere found in roughly equal ratios in both the PC pool and in TAG. Therewere slight variations in this ratio for the products of the second laststep of the pathways, a Δ5-elongation producing DTA and DPA, which wereless abundant on TAG compared to PC. Conversely, the products of thefinal Δ4 desaturation, DPA^(ω6) and DHA, were preferentially accumulatedon TAG compared to PC. These variations may reflect subtle biases of themembrane editing enzymes or the AtDGAT1 for these products. It is worthnoting that both Δ5-elongations and DGAT activities occur on CoA pools,and competition between these enzymes for substrate may alter thepresence of fatty acids in these PC and TAG pools.

There are several implications that arise from this study. First, thetransient leaf based assay was shown to be suitable for assaying fattyacid enzymes, either singly or in complex combinations. This isparticularly apt for enzymes producing fatty acids that are easilydistinguished from the endogenous fatty acid profile of N. benthamiana,such as the LC-PUFA in this study. The demonstration of fattyacid-feeding assays to isolated enzymes and the rapid assembly ofLC-PUFA into oils indicated that the transient leaf assay was wellsuited for ER-associated desaturation, elongation and TAG assembly.Second, although LC-PUFA oils are a current target of planttransformation technologies, leaf cells provide a range of advantages toother heterologous expression platforms. Leaf cells provide a wide rangeof metabolites not available in other expression hosts, and these maynow become targets for modification requiring recombinant pathways.Furthermore plants more faithfully process eukaryotic transgenes,including RNA editing, post-translation modifications and organellelocalisation.

Finally, the N. benthamiana assay could be suitable for cDNA libraryscreening assays. This suggestion is based on the detection ofnear-maximal activity for seven different genes in a single infiltrationzone on the leaf, suggesting that in this configuration cDNA librariescloned within a binary expression vector could be systematicallyinfiltrated into leaves. Calculations suggest that at least 7 differentclones, including P19 at OD_(600nm)0.2, could be expressed in a singlespot. Alternatively, genes forming incomplete or partial pathways couldbe added to each infiltration and thereby pooled library clones could beroutinely tested for novel steps or improved fluxes.

Example 10 Efficient DHA Biosynthesis in Plant Cells

The enzyme activities of the Micromonas CCMP1545 Δ6-desaturase (SEQ IDNO:8 encoded by SEQ ID NO:7), Pyramimonas CS-0140 Δ6-elongase (SEQ IDNO:4 encoded by SEQ ID NO:3), Pavlova salina Δ5-desaturase (SEQ ID NO:26encoded by SEQ ID NO:25), Pyramimonas CS-0140 Δ5-elongase (SEQ ID NO:6encoded by SEQ ID NO:5) and Pavlova salina Δ4-desaturase (SEQ ID NO:73encoded by SEQ ID NO:72) along with the Arabidopsis thaliana DGAT1 (SEQID NO:74 encoded by SEQ ID NO:75) were demonstrated in planta using anenhanced Nicotiana benthamiana transient expression system as describedin Example 1.

A genetic construct 35S:Mic1545-d6D encoding the Δ6-desaturase under thecontrol of the constitutive 35S promoter was made by inserting theentire coding region of pGA4, contained within a SwaI fragment, into35S-pORE04 (Example 4, above) at the SmaI-EcoRV site, generatingpJP2064. A genetic construct 35S:Pyrco-d6E encoding the Δ6-elongase wasmade by inserting the entire coding region of 0804673_Pyrco-elo1_pGA18,contained within a SwaI fragment, into 35S-pORE04 at the SmaI-EcoRVsite, generating pJP2060. A genetic construct 35S:Pavsa-d5D encoding theΔ5-desaturase was made by inserting the entire coding region of0804674_Pavsa-d5D_pGA15, contained within a SwaI fragment, into35S-pORE04 at the SmaI-EcoRV site, generating pJP2067. A geneticconstruct 35S:Pyrco-d5E encoding the Δ5-elongase was made by insertingthe entire coding region of 0804675 Pyrco-elo2 pGA4, contained within aSwaI fragment, into 35S-pORE04 at the SmaI-EcoRV site, generatingpJP2061. A genetic construct 35S:Pavsa-d4D encoding the Δ4-desaturasewas made by inserting the entire coding region of0804676_Pavsa-d4D_pGA15, contained within a SwaI fragment, into35S-pORE04 at the SmaI-EcoRV site, generating pJP2068. A geneticconstruct 35S:Arath-DGAT1 encoding the enzyme DGAT1 was made byinserting the entire coding region of pXZP513E, contained within aBamHI-EcoRV fragment, into 35S-pORE04 at the BamHI-EcoRV site,generating pJP2078.

These chimeric vectors were introduced individually into Agrobacteriumtumefaciens strain AGL1 and transgenic cells from cultures of these weremixed and the mixture infiltrated into leaf tissue of Nicotianabenthamiana plants in the greenhouse. The plants were grown for afurther five days after infiltration before leaf discs were taken for GCanalysis which revealed that these genes were functioning to produce DHAin Nicotiana benthamiana. Leaf tissue transformed with these genescontained SDA (2.3%), ETA (0.7%), EPA (0.8%), DPA (2.8%) and DHA (4.4%)(Table 14). The leaf tissue also contained trace levels of GLA, ETA andARA. The conversion efficiencies were as follows: 17.4% of the ALAproduced in the cell was converted to EPA (including EPA subsequentlyconverted to DPA or DHA); 15.5% of ALA was converted to DPA or DHA; 9.6%of the ALA produced in the cell was converted to DHA; while 40% of theALA produced in the cell that was Δ6-desaturated was subsequentlyconverted to DHA. Due to the transient expression of the transgenes inthis experiment, higher efficiencies than the above would be expected instably transformed cells.

When the total lipid extracted from the leaf tissue was fractionated byTLC to separate the lipid classes, and the TAG and polar lipid fractionsanalysed for fatty acid composition by FAME, it was observed that thelevel of DHA in the TAG was 7% as a percentage of the total fatty acid,and in the polar-lipid the level of DHA was 2.8%. The lower level in thepolar lipid class was thought to be due to the relative contribution ofchloroplast lipids in the leaves, favouring polar-lipids, and thetransient expression of the genes rather than stable insertion of thetransgenes into the host cell genome.

TABLE 14 Fatty acid composition of lipid from leaves transformed with acombination of desaturases and elongases. Fatty acid Fatty acidUntransformed cells Transformed cells 16:0 palmitic 17.1 20.4 16:1d7 0.10.4 16:1d9 0.2 0.2 C6:1d? 0.5 0.4 16:1d? 0.5 0.4 17:1d9 0.9 0.7 16:2 0.90.9 16:3 6.6 5.4 18:0 stearic 2.1 3.6 18:1d7 0.0 0.0 18:1d9 oleic 0.82.6 18:1d11 0.0 0.0 18:1d12 0.3 0.6 18:1d13 0.2 0.2 18:2n6 LA 5.1 10.818:3n6 GLA 0.7 1.9 18:3n3 ALA 57.8 35.0 20:0 0.4 0.7 20:1d5 0.4 0.318:4n3 SDA 0.4 2.3 20:1d8 0.0 0.0 20:1d11 0.0 0.0 20:2n6 EDA 0.1 0.220:3n6 DGLA 0.3 0.4 20:4n6 ARA 0.4 0.5 20:3n3 ETrA 0.2 0.2 22:0 0.2 0.220:4n3 ETA 0.0 0.7 22:1d9 0.0 0.9 20:5n3 EPA 0.8 0.8 22:2n6 0.1 0.222:4n6 0.1 0.1 22:3n3 1.1 1.2 24:0 0.4 0.4 22:5n6 0.4 0.2 24:1d9 0.8 0.122:5n3 DPA 0.2 2.8 22:6n3 DHA 0.1 4.4

This experiment demonstrated that the isolated Micromonas CCMP1545Δ6-desaturase had a substantial preference for the ω3 substrate ALAcompared with the ω6 substrate LA. The experiment also demonstrated thatthe expression of suitable genes can result in the accumulation ofsubstantial percentages of LC-PUFA in the leaf, most notably EPA, DPAand DHA.

This experiment also showed the use the Nicotiana benthamiana transientassay system for the rapid testing of various fatty acid biosynthesispathways and for selecting optimal combinations of genes. This systemcould be used to rapidly compare the relative activities of genes withhomologous function, as well as the comparison of entire biosyntheticpathways.

Discussion: Efficient DHA Synthesis in Leaf and Seed Tissue

On the basis of this data, it was predicted that the same levels of EPA,DPA and DHA, or even higher levels, would be produced in seed whenseed-specific promoters are used to express this combination of genes,or a similar set. The observed efficient flux of fatty acids from ALA toEPA and through to DPA and DHA was thought to be due to the combinationof efficient elongases with acyl-CoA desaturases, thereby operating onfatty acids predominantly in the acyl-CoA pool. Furthermore, it waspredicted that production of EPA, DPA, DHA and other LC-PUFA in bothleaf and seed of a transgenic plant, or in seed and another tissue otherthan leaves, could be achieved by the use of a promoters with theappropriate tissue specificity, or promoter combinations. Fusedpromoters would be able to drive the production of the enzymes in bothtissue types. The resulting plant would likely be useful for both oilextraction, particularly from seed, and feedstock with minimalprocessing.

Example 11 More Efficient DHA Biosynthesis in Plant Cells

The enzyme activities of the Micromonas CCMP1545 Δ6-desaturase (SEQ IDNO:8 encoded by SEQ ID NO:7), Pyramimonas CS-0140 Δ6-elongase (SEQ IDNO:4 encoded by SEQ ID NO:3), Pavlova salina Δ5-desaturase (SEQ ID NO:26encoded by SEQ ID NO:25), Pyramimonas CS-0140 Δ5-elongase (SEQ ID NO:6encoded by SEQ ID NO:5) and Pavlova salina Δ4-desaturase (SEQ ID NO:73encoded by SEQ ID NO:72) along with the Arabidopsis thaliana DGAT1 (SEQID NO:74 encoded by SEQ ID NO:75) were demonstrated in planta using anenhanced Nicotiana benthamiana transient expression system as describedin Example 1 and Example 10. This experiment was optimised by usingyounger, healthier N. benthamiana plants.

These chimeric vectors described in Example 10 were introducedindividually into Agrobacterium tumefaciens strain AGL1 and transgeniccells from cultures of these were mixed and the mixture infiltrated intoleaf tissue of Nicotiana benthamiana plants in the greenhouse. Theplants were grown for a further five days after infiltration before leafdiscs were taken for GC analysis which revealed that these genes werefunctioning to produce DHA in Nicotiana benthamiana (Tables 15 and 16).Leaf tissue transformed with these genes contained SDA (2.0%), ETA(0.4%), EPA (0.7%), DPA (4.3%) and DHA (4.4%). The leaf tissue alsocontained trace levels of GLA, ETA and ARA. The conversion efficiencieswere as follows: 23.4% of the ALA produced in the cell was converted toEPA (including EPA subsequently converted to DPA or DHA); 21.6% of ALAwas converted to DPA or DHA; 10.9% of the ALA produced in the cell wasconverted to DHA; while 37.2% of the ALA produced in the cell that wasD6-desaturated was subsequently converted to DHA. Due to the transientexpression of the transgenes in this experiment, higher efficienciesthan the above would be expected in stably transformed cells.

When the total lipid extracted from the leaf tissue was fractionated byTLC to separate the lipid classes, and the TAG and polar lipid fractionsanalysed for fatty acid composition by FAME, it was observed that thelevel of DHA in the TAG was 15.9% as a percentage of the total fattyacid, and in the polar-lipid the level of DHA was 4.4%. The lower levelin the polar lipid class was thought to be due to the relativecontribution of chloroplast lipids in the leaves, favouringpolar-lipids, and the transient expression of the genes rather thanstable insertion of the transgenes into the host cell genome.

TABLE 15 Fatty acid composition of lipid from leaves transformed with acombination of desaturases and elongases. Control Pavsa-D5E Pyrco-D5ETotal DHA Pathway DHA Pathway Fatty acid Lipid Total Lipid TAG TotalLipid TAG Usual FA 16:0 15.9 ± 0.2  17.0 ± 0.1  20.2 16.6 ± 0.1  21.616:1^(Δ3t) 1.7 ± 0.1 1.5 ± 0.2 0.3 1.5 ± 0.1 0.3 16:3^(Δ9,12,15) 6.3 ±0.3 5.2 ± 0.1 0.4 5.6 ± 0.1 0.6 18:0 3.6 ± 0.3 3.5 ± 0.1 5.9 3.3 ± 0.16.5 18:1^(Δ9) 2.8 ± 0.1 3.4 ± 0.1 5.1 2.8 ± 0.2 5.6 18:2^(Δ9,12) 18.7 ±0.1  14.1 ± 0.4  15.2 13.0 ± 0.1  17.3 18:3^(Δ9,12,15) 45.6 ± 1.4  39.1± 0.4  6.9 40.2 ± 0.5  6.7 20:0 1.3 ± 0.4 0.7 ± 0   1.8 0.6 ± 0   2.0Other minor   4.1  2.5 6.3 2.3 6.2 Total 100  87.0 62.1 85.9  66.8 Neww6 PUFA 18:3^(Δ6,9,12) — 1.6 ± 0.1 3.3 2.1 ± 0.2 4.3 20:3^(Δ8,11,14) — —— — — 20:4^(Δ5,8,11,14) — 0.3 ± 0.1 0.2 ± 0   — Total 0  1.9 3.8 2.3 4.3New ω3 PUFA 18:4^(Δ6,9,12,15) — 1.5 ± 0.1 3.8 2.0 ± 0   6.4 (22% D6D)(23% D6D) 20:4^(Δ8,11,14,17) — 0.5 ± 0   1.5 0.4 ± 0   1.6 (86% D6E)(83% D6E) 20:5^(Δ5,8,11,14,17) — 4.1 ± 0.2 14.2 0.7 ± 0   1.5 (95% D5D)(96% D5D) 22:5^(Δ7,10,13,16,19) — 2.4 ± 0.1 1.6 4.3 ± 0   3.5 (55% D5E(93% D5E) 22:6^(Δ4,7,10,13,16,19) — 2.6 ± 0.1 13.0 4.4 ± 0.1 15.9 (52%D4D) (51% D4D) Total 0 11.1 34.1 11.8  28.9 Total new FA 0 13.0 37.914.0  33.2 Total FA 100  100   100 100    100

TABLE 16 Conversion efficiencies from leaves transformed with acombination of desaturases and elongases. Fatty Acid Enzyme ConverionEfficiency Total LA 13 GLA 2.1 15.0% d6D DGLA 0 8.7% d6E AA 0.2 100.0%d5D ALA 40.2 SDA 2 22.7% d6D 18.1% d6D to EPA + DPA + DHA ETA 0.4 83.1%d6E 16.7% d6D to DPA + DHA EPA 0.7 95.9% d5D 8.5% ALA to DHA DPA 4.392.6% d5E 46.8% EPA to DHA DHA 4.4 50.6% d4D TAG LA 17.3 GLA 4.3 19.9%d6D DGLA 0 0.0% d6E AA 0 0.0% d5D ALA 6.7 SDA 6.4 81.2% d6D 58.7% d6D toEPA + DPA + DHA ETA 1.6 77.9% d6E 54.5% d6D to DPA + DIIA EPA 1.5 92.9%d5D 44.7% ALA to DHA DPA 3.5 92.8% d5E 76.1% EPA to DHA DHA 15.9 82.0%d4DDiscussion: More Efficient DHA Synthesis in Leaf and Seed Tissue

This result is likely to pave the way for similar advances in yield inseed TAG due to the substantial conservation of extra-plastidial lipidsynthesis mechanisms between leaf and seed tissues (Ohlrogge and Browse,2004; Bates et al., 2007). We postulate that several elements are likelyresponsible for this large increase in production: 1. the use of anω3-specific acyl-CoA Δ6-desaturase increases flux down the ω3 pathwayand decreases competition with parallel ω6 substrates for subsequentmetabolic steps; 2. a highly efficient Δ5-elongase clearly increases theamount of DPA available for Δ4-desaturation to DHA; 3. the reduction ofgene silencing by the use of independent transcriptional units and theuse of a viral suppressor protein (P19).

The strong ω3 preference displayed by the Δ6-desaturase is clearlydesirable when attempting to engineer a land plant that accumulates theω3 LC-PUFA EPA and DHA since the additional Δ17-desaturase activityrequired to convert AA (20:4^(D5,8,11,14)) to EPA is not required, thussimplifying both metabolic engineering and regulatory challenges.

In addition to the above optimised step, use of the highly efficient P.cordata Δ5-elongase resulted in a fatty acid profile of the TAG (oil)fraction that closely resembled tuna oil, a fish oil notable for highDHA and low intermediate content (FIG. 18). Also, the activity displayedby the P. cordata Δ5-elongase in N. benthamiana is by far the mostefficient Δ5-elongation we have experienced and use of this geneeffectively overcomes the large Δ5-elongation bottleneck that has beenexperienced in other attempts at transgenic DHA production.

Finally, whilst the use of optimal genes is clearly required we considerit probable that the method by which these transgenes were introduced(i.e. as independent expression cassettes and in the presence of agene-silencing suppressor) played a key role in the high levels of DHAachieved in this study. Metabolic engineering for LC-PUFA production hasthus far relied on relatively large multi-gene constructs being randomlyinserted into the host genome and whilst many groups have had goodresults with this method there are indications that it is difficult toobtain events displaying equal expression of all the transgenes (WO2004/017467). In addition, silencing effects may reduce efficiency overgenerations (Matzke et al., 2001). It is possible that alternativetransformation approaches such as artificial chromosomes involving denovo centromere formation on an independently assembled unit andengineered mini-chromosomes might ultimately be required for successfulstable LC-PUFA metabolic engineering (Yu et al., 2007).

Example 12 Transgenic Assembly of an Entire ALA to DHA Pathway UsingGenes from a Single Organism

The entire ALA to DHA pathway was reconstituted using genes encoding theenzymes from P. salina, consisting of the Δ9-elongase, Δ8-desaturase,Δ5-desaturase, Δ5-elongase and Δ4-desaturase, and assembled in N.benthamiana. GC analysis of the total leaf tissue five days afteragroinfiltration demonstrated the production of 0.7% DHA (Table 17).This is the first time a transgenic pathway from ALA to DHA consistingof genes from a single organism has been reported.

TABLE 17 Fatty acid composition (percent of total fatty acids) ofNicotiana benthamiana leaf tissue transiently transformed withsingle-gene CaMV 35S binary constructs. The DHA pathway consists of theP. salina Δ9-elongase, Δ8-desaturase, Δ5-desaturase, Δ5-elongase andΔ4-desaturase. Standard deviations between separate infiltrationsperformed in triplicate are indicated. Fatty acid Control Pavpi-Δ9E DHAUsual FA 16:0 15.7 ± 0.6 15.9 ± 0.2  16:1^(Δ3t) 1.5 ± 0  1.4 ± 0.116:3^(Δ9,12,15)  6.8 ± 0.7 5.9 ± 0.3 18:0  3.0 ± 0.1 3.7 ± 0.2 18:1^(Δ9)2.2 ± 0  2.7 ± 0.3 18:2^(Δ9,12) 11.8 ± 0.4 8.6 ± 0.6 18:3^(Δ9,12,15)56.0 ± 1.4 49.0 ± 1.4  Other minor 3.0 ± 0  2.8 ± 0   Total 100  90.0New ω6 PUFA 20:2^(Δ8,11) — 1.7 ± 0.2 20:3^(Δ8,11,14) — 0.5 ± 0  20:4^(Δ5,8,11,14) — 2.4 ± 0.1 22:4^(Δ7,10,13,16) — 1.2 ± 0  22:5^(Δ4,7,10,13,16) — — Total 0  5.8 New ω3 PUFA 20:3^(Δ11,14,17) — 1.5± 0.2 20:4^(Δ8,11,14,17) — 0.2 ± 0   20:5^(Δ5,8,11,14,17) — 1.2 ± 0.122:5^(Δ7,10,13,16,19) — 0.6 ± 0   22:6^(Δ4,7,10,13,16,19) — 0.7 ± 0.1Total 0  4.2 Total new FA 0 10.0 Total FA 100  100  

Example 13 Specific Expression of VSPs in Developing Seeds of Plants

The protein coding regions of five viral suppressor proteins (VSP),namely P19, V2, P38, PePo and RPV-P0, were initially inserted into abinary vector pART27 (Gleave, 1992) under the control of the 35Spromoter for strong constitutive expression in plant tissues. Theseproteins have been characterised as VSPs as follows. P19 is a suppressorprotein from Tomato Bushy Stunt Virus (TBSV) which binds to 21nucleotide long siRNAs before they guide Argonaute-guided cleavage ofhomologous RNA (Voinnet et al., 2003). V2, a suppressor protein fromTomato Yellow Leaf Roll Virus (TYLRV), binds to the plant protein SGS3(Glick et al., 2008), a protein thought to be required for theproduction of double stranded RNA intermediates from ssRNA substrates(Beclin et al., 2002). P38 is a suppressor protein from Turnip CrinkleVirus (TCV) and inhibits the RNA dependent polymerase activity (RdRP)critical for the production of siRNA and binds to the Dicer protein DCL4(Ding and Voinnet, 2007). P0 proteins such as PePo and RPV-P0, frompoleroviruses, target Argonaut proteins for enhanced degradation(Baumberger et al., 2007; Bortolamiol et al., 2007). In order toestablish the function of these proteins to increase transgeneexpression as suppressors of silencing, the five 35S driven VSPconstructs in Agrobacterium were co-infiltrated with a 35S-driven GFPconstruct into Nicotiana benthamiana leaves. In all cases the presenceof the VSP increased and extended expression of the GFP, conferringincreased levels of GFP gene activity particularly after 4 dayspost-inoculation with the Agrobacterium strains, confirming the functionof the proteins as silencing suppressors in this assay format.

The five VSP coding regions were each inserted into a second binaryvector, pXRZ393, based on a pART27 backbone vector, so that expressionof the VSPs was under the control of a seed-specific FP1 promoter(Ellerstrom et al., 1996) providing expression of the VSPs in cotyledonsof developing seed in dicotyledonous plants. The constructs were used togenerate transformed Arabidopsis plants according to the methodsdescribed in Example 1. At least 20 transformed plants were obtained foreach chimeric gene encoding VSP. The plants were viable andphenotypically mostly normal as indicated by their growth on selectionmedia and in soil, growing normally into fertile adult plants which setviable seed. The only morphological phenotype that appeared to bealtered in the seedlings was in the cotyledons that emerged from some ofthe seeds after germination for P19, PePo and RPV-P0. Plantletstransformed with the construct seed-specifically encoding P19 had flatclub-shaped cotyledons with no downward curl distinctive of wild-typeplantlets. Plantlets transformed with the construct encoding PePoproduced a ‘ballerina’ phenotype, where the cotyledons were up-wardpointing with an inward or concave curl. True leaves on these plantswere normally developed. Plantlets expressing RPV-P0 were bushy, andtended to retain a bushy growth habit throughout vegetative growth.Plantlets of V2 and P38 displayed no apparent, significant phenotype.

Other than cotyledon development, plantlets transformed with the P19 andPoPe constructs grew normally and were indistinguishable from controlplants in subsequent growth and development. The promoter expressing theVSPs in this experiment, FP1, is well characterised with a limited butstrong expression in developing cotyledons of Arabidopsis during seeddevelopment (Ellerstrom et al., 1996). On the basis of the emergentcotyledon phenotypes, we suggest that FP1-driven expression of P19, PePoor RPV-P0 in developing seed may overlap with small RNA biogenesisrequired for normal cotyledon development. These VSP-related changes incotyledon development do not impact on the overall development of thetransgenic plants, and the normal subsequent growth and development ofthe plants indicated that any leaky expression of VSPs from the FP1promoter in tissues other than the developing seed was minor andinsignificant. This was in contrast to previous studies whereconstitutive expression of many VSPs in plant tissues was highlydeleterious (Mallory et al., 2002; Chapman et al., 2004; Chen et al.,2004; Dunoyer et al., 2004; Zhang et al., 2006; Lewsey et al., 2007:Meng et al., 2008).

The lack of a phenotype for the VSPs V2 and P38 may reflect that theseVSPs target small RNA metabolism in ways that do not affectdevelopmental small RNA biogenesis or recognition. The functionality ofeach VSP was checked using a GFP assay in Nicotiana benthamiana.

Example 14 Development of a Seed-specific Visual Marker to Find andAssess T1 Seed Transformed with VSP Constructs

The data described in Example 13 indicated that seeds, and thesubsequent progeny plants, could tolerate the expression of VSPs fromseed-specific promoters without significant deleterious effects. Theinventors also considered whether the transformed plants generated asdescribed above were expressing only low levels of the VSPs, therebyallowing the seed to survive, effectively selecting against transformedseeds potentially expressing lethal amounts of VSP which would thereforenot be recovered under the conditions used.

To more accurately assess the expression of transgenes in T1 seed, avisual marker of transformation and expression was developed for use intransgenic seed. Arabidopsis seeds as for other dicot seeds contain apaternal embryo and endosperm surrounded by a maternal seed coat. DuringAgrobacterium-mediated transformation of Arabidopsis, paternal tissuebecomes transformed by the T-DNA whilst the maternal tissue remainsuntransformed. Transformed maternal tissue is not obtained until theproduction of T2 seed of the next generation. To provide a usefulscreening system, a gene encoding green fluorescent protein (GFP) wasmodified to promote strong secretion of the protein outside of the cell.Such a location of GFP had been show to allow visual recovery oftransformed T1 seed (Nishizawa et al., 2003) by detection of thesecreted GFP produced in the transformed T1 embryo and endosperm,through the thin but untransformed seed coat (Fuji et al., 2007).

A chimeric gene encoding a secreted GFP was constructed as follows. Thegene contained two introns, one in the 5′untranslated region (5′UTR) anda second in the protein coding region of GFP. These introns wereincluded to enhance expression of the chimeric gene (Chung et al., 2006)and to ensure that any GFP signal detected in the seeds could onlyresult from expression of the gene in plant cells rather than leakyexpression from the Agrobacterium cells. An intron-interrupted versionof humanised GFP (Clontech) encoding a GFP protein that would belocalised to the cytoplasm (Brosnan et al., 2007) was modified topromote secretion to the apoplast via the endoplasmic reticulum (ER)according to the report of Nishizawa et al. (2003). The modified GFPconstruct for apoplastic secretion included a conglycinin secretionpeptide added as an in-frame translational fusion at the N-terminus ofGFP and four glycine residues added at the C-terminus to facilitatesecretion from the ER. These additions to the chimeric GFP gene wereperformed by gene synthesis for the 5′ region and PCR-mediated sequencemodifications for the C-terminal glycine additions. The coding regionfor this secreted GFP was inserted downstream of a FP1 promoter whichhad an intron from a catalase gene in the 5′UTR. The entire FP1-secretedGFP sequence was inserted upstream of a nos3′ polyadenylation signalwithin a pORE series binary vector. The construct containing thechimeric gene encoding the secreted GFP was designated pCW141.

To confirm the expression and secretion of the GFP protein, the generegion encoding the secreted GFP sequence but without the FP1 promoteror the 5′UTR of pCW141 was subcloned into a pCaMV35S-OCS3′ expressioncassette, to produce pCW228, and introduced by Agrobacterium-mediatedtransformation into N. benthamiana leaves. Expression of the gene andsecretion of GFP protein was confirmed using confocal microscopy(Leica).

On the basis that the construct was correct, pCW141 was introduced intoplants of Arabidopsis Col-0 ecotype via Agrobacteria-mediated methods asdescribed in Example 1. Seeds from Arabidopsis plants dipped withpCW141, the FP1-driven secreted GFP construct, were collected andscreened for GFP-positive seeds using a dissecting microscope equippedfor fluorescence detection (Leica MFZIII). Seeds that fluoresced greenwere easily identified even though the vast majority of T1 seeds in thepopulations were untransformed. These GFP-positive seeds were selectedand grown on selective media to confirm the presence of thekanamycin-resistance selectable marker gene linked to the chimeric geneconstruct on the T-DNA. A further 20 positive seed were pooled and theexpression of GFP protein was confirmed by Western Blotting using anantibody against GFP.

Selection of VSP Expression in T1 Seed Using a Secreted GFP Marker

Each of the four chimeric genes encoding the VSPs: P19, P38, V2 and P38,each under the control of the FP1 promoter for developingcotyledon-specific expression, were inserted into the GFP selectionvector pCW141 described above to produce pCW161, pCW162, pCW163 andpCW164, respectively. These binary vectors each had linked chimericgenes for the expression of a VSP and the secreted GFP, and therebyallowed transgenic seed transformed with the constructs to beidentified, selected and analysed by the GFP phenotype without growth onselective media. It was expected that in the majority of transformants,the gene encoding the VSP would be integrated and therefore linked withthe gene encoding GFP.

Seeds obtained from Arabidopsis plants which had been inoculated withAgrobacterium containing the combination VSP-GFP constructs, accordingto the method in Example 1, were collected and screened for GFPfluorescence as described above. Seeds that fluoresced green werecollected by hand and grown on selective media to determine whether theyhad also been transformed with the selectable marker gene. In all casesthe GFP-positive seed grew on the selection media, and exhibited thesame cotyledon phenotypes as plants that had been transformed with theVSP genes without the GFP gene as described above. In no cases were GFPpositive seed observed that failed to grow on the selective media. Suchseeds might have been expected if some transformation events gave riseto cells in the developing seeds in which the expression levels of VSPwas too high and caused lethality. The absence of such seeds in thetransformed populations indicated that the VSP expression was toleratedin seeds when expressed from the FP1 promoter. The absence of suchdeleterious effects was in contrast to reports of deleterious effectswhen many VSPs were expressed constitutively (Mallory et al., 2002;Chapman et al., 2004; Chen et al., 2004; Dunoyer et al., 2004; Zhang etal., 2006; Lewsey et al., 2007: Meng et al., 2008).

Thus, use of a visually-detectable marker such as GFP proved a powerfuland efficient way to identify, select and analyse transgenic eventsincorporating linked genes such as those encoding VSPs, desaturase,elongases or other fatty acid-modifying enzymes.

Quantification of GFP Expression in Seeds Expressing VSPPost-embryonically

GFP expression was quantified in T1 and T2 seed expressing VSP from FP1promoter using fluorescent microscopy and digital image analysis. Theseanalyses clearly showed that GFP expression was not affected by theco-introduction of a gene encoding VSP under the control of thedeveloping cotyledon-specific promoter. Extended studies on theperformance of GFP-VSP constructs over subsequent generations and inindependent transformation events will be analysed. It is predicted thatthe presence of the VSP will result in more stable and higher averagelevels of expression in successive generations of seed.

Example 15 Co-expression of VSPs with Genes for LC-PUFA Synthesis inSeeds

To establish that VSPs are capable of protecting or enhancing transgeneperformance in seeds, some expression vectors were designed that wereconsidered to be more prone to host-mediated suppression (silencing)than a vector with only a single gene, to increase the relativeeffectiveness of the VSPs. A series of vectors each containing fivedesaturase or elongase genes for DHA synthesis in seeds wereconstructed, using the same configuration of the genes in each. Onefactor that was thought to make these constructs more prone to silencing(reduced expression) was the use of the same promoter (FP1) to driveeach gene. The FP1 promoter was used as it was relatively small andreduced the overall vector size and the spacing between each codingregion. Furthermore, each gene cassette had the same orientation, whichwe considered would enhance the likelihood of silencing. Three genes ofthe LC-PUFA pathway had coding regions which had been codon-optimised(A-B-C) for optimal plant expression while two (E-D) were nativesequences as obtained from the microalgae. The same suite of five geneshad previously been expressed in leaves to produce assemble a completeLC-PUFA biosynthetic pathway (Example 11). A further gene encoding theVSP P19 was included in the first vector of the series, a gene encodingV2 was included in the second vector, while the third vector in theseries had no VSP.

These vectors pJP3057 (FIG. 19), pJP3059 (FIG. 20) and (FIG. 21) wereconstructed and transformed in parallel as follows. Desaturase orelongase genes or viral suppressor genes were first cloned between a FP1promoter and nos terminator contained within a cloning vector. Thepromoter-gene-terminator cassettes were then cloned sequentially and inthe same orientation into a binary vector backbone. pJP3057 containedthe entire DHA pathway whilst pJP3059 and pJP3060 differed only in theaddition of a FP1-P19-NOS or FP1-V2-NOS cassette, respectively. Theconstruction steps were as follows. First, an AscI-PacI fragmentcontaining the Micromonas pusilla Δ6-desaturase was cloned into theAscI-PacI site of pJP2015 before a SwaI fragment from this vectorcontaining the entire promoter-gene-terminator cassette was cloned intothe EcoRV site of pORE02 to generate pJP3050. Next, an AscI-PacIfragment containing the Pyramimonas cordata Δ6-elongase was cloned intothe AscI-PacI site of pJP2015TMV (a slightly modified version of pJP2015where the TMV leader was present downstream of the promoter and upstreamof the gene) before a SwaI fragment from this vector containing theentire promoter-gene-terminator cassette was cloned into the T4 DNApolymerase-treated SacI site of pJP3050 to generate pJP3051. Next, anAscI-PacI fragment containing the Pavlova salina Δ5-desaturase wascloned into the AscI-PacI site of pJP2015TMV before a SwaI fragment fromthis vector containing the entire promoter-gene-terminator cassette wascloned into the SmaI site of pJP3051 to generate pJP3052. Next, anAscI-PacI fragment containing the Pavlova salina Δ5-desaturase wascloned into the AscI-PacI site of pJP2015TMV before a SwaI fragment fromthis vector containing the entire promoter-gene-terminator cassette wascloned into the SmaI site of pORE02 to generate pJP3054. Next, anAscI-PacI fragment containing the Pyramimonas cordata Δ5-elongase wascloned into the AscI-PacI site of pJP2015TMV before a SwaI fragment fromthis vector containing the entire promoter-gene-terminator cassette wascloned into the StuI site of pJP3054 to generate pJP3055. Next, anAscI-PacI fragment containing the Pavlova salina Δ4-desaturase wascloned into the AscI-PacI site of pJP2015TMV before a SwaI fragment fromthis vector containing the entire promoter-gene-terminator cassette wascloned into the SfoI site of pJP3056 to generate pJP3056. The PmeI-NotIfragment of pJP3056 was then cloned into the PmeI-NotI site of pJP3051to generate pJP3057, a binary vector containing the five genes forproduction of DHA from ALA.

Next, an AscI-PacI fragment containing the chimeric gene encoding theP19 viral suppressor was cloned into the AscI-PacI site of pJP2015TMVbefore a SwaI fragment from this vector containing the entirepromoter-gene-terminator cassette was cloned into the ZraI site ofpJP3057 to generate pJP3059. Similarly, an AscI-PacI fragment containingthe chimeric gene encoding the V2 viral suppressor was cloned into theAscI-PacI site of pJP2015TMV before a SwaI fragment from this vectorcontaining the entire promoter-gene-terminator cassette was cloned intothe ZraI site of pJP3057 to generate pJP3060.

All three constructs were transformed in Arabidopsis (ecotype Columbia).Arabidopsis plants (Col-0 ecotype) were transformed with each of theconstructs and pJP3057 was used to transform canola. T1 seeds will becollected, analysed on herbicide-containing media, and the resulting T2seed analysed for general morphological changes and LC-PUFA synthesis.

The transformed plants (Arabidopsis thaliana, ecotype Columbia)generated with the three constructs pJP3057, pJP3059 and pJP3060 wereself-fertilised and T1 seeds were collected. These were sown onkanamycin-containing media to determine heterozygosity/homozygosity ofthe T1 plants, and the resultant T2 seed from each of the T1 plants wereanalysed for general morphological changes and LC-PUFA synthesis (Table18).

Representative T2 seed of plants transformed with pJP3057 contained, inthe total fatty acid in the seedoil, SDA (0.4%), ETA (0.6%), EPA (0.2%),DPA (0.3%) and DHA (2.4%). The seedoil of the T2 plants also containedGLA (1.4%) and trace levels of ETrA and ARA. The conversion efficienciesin the seed were as follows: 18.4% of the ALA produced in the cell wasΔ6-desaturated; 89.7% of the SDA produced in the cell was Δ6-elongated;82.9% of the ETA in the cell was Δ5-desaturated; 93.1% of the EPA in thecell was Δ5-elongated; 88.9% of the DPA in the cell was Δ4-desaturatedto produce DHA (Table 18).

Representative T2 seed of plants transformed with pJP3059 contained SDA(0.7%), ETA (0.3%), EPA (0.2%), DPA (0.9%) and DHA (1.3%). The seedoilalso contained GLA (0.8%) and trace levels of ETrA and ARA. Theconversion efficiencies were as follows: 15.7% of the ALA produced inthe cell was Δ6-desaturated; 79.4% of the SDA produced in the cell wasΔ6-elongated; 88.9% of the ETA in the cell was Δ5-desaturated; 91.7% ofthe EPA in the cell was Δ5-elongated; 59.1% of the DPA in the cell wasΔ4-desaturated to produce DHA (Table 18).

TABLE 18 Representative fatty acid profiles of T2 Arabidopsis seedstransformed with pJP3057, pJP3059, pJP3060. Sample Columbia pJP3057pJP3059 pJP3060 16:0 7.7 7.6 8.3 7.4 16:1d9 0.3 0.3 0.3 0.3 18:0 3.1 3.73.8 3.4 20:0 2.1 1.8 1.8 1.9 22:0 0.3 0.3 0.3 0.3 24:0 0.2 0.2 0.2 0.218:1d9 12.9 12.8 12.2 13.6 18:1d11 1.5 1.8 1.9 1.6 20:1d11 18.3 16.314.7 16.0 20:1d13 1.7 1.5 2.0 1.9 22:1d13 1.6 1.2 1.2 1.3 24:1d15 0.20.2 0.2 0.2 Other 2.5 2.3 2.6 2.5 18:2n6 27.8 27.2 28.2 27.9 18:3n6 0.01.4 0.8 0.4 20:3n6 0.0 0.0 0.0 0.0 20:4n6 0.0 0.0 0.0 0.0 18:3n3 19.717.3 18.2 18.2 18:4n3 0.0 0.4 0.7 0.6 20:4n3 0.0 0.6 0.3 0.7 20:5n3 0.00.2 0.2 0.3 22:5n3 0.0 0.3 0.9 0.2 22:6n3 0.0 2.4 1.3 1.0 100.0 100.0100.0 100.0

Representative T2 seed of plants transformed with pJP3060 contained SDA(0.6%), ETA (0.7%), EPA (0.3%), DPA (0.2%) and DHA (1.0%). The seedoilalso contained trace levels of GLA, ETrA and ARA. The conversionefficiencies were as follows: 13.3% of the ALA produced in the cell wasΔ6-desaturated; 78.6% of the SDA produced in the cell was Δ6-elongated;68.2% of the ETA in the cell was Δ5-desaturated; 80.0% of the EPA in thecell was Δ5-elongated; 83.3% of the DPA in the cell was Δ4-desaturatedto produce DHA (Table 18).

Results

All genes in the construct pJP3057 showed high activity/high efficiencyof conversion with the exception of the Δ6-desaturase. This indicatesthat the Δ6-, Δ5- and Δ4-desaturases are likely acting on acyl-CoAsubstrates since the native substrate ALA is produced by an acyl-PCdesaturase, resulting in the lower Δ6-desaturation, and the transgenicdesaturase substrates ETA and DPA are produced by elongases which areknown to act in the acyl-CoA pool. Furthermore, the high efficiency ofthe Δ6- and Δ5-elongase steps (>80% efficiency) indicated that theimmediately preceding desaturases (Δ6- and Δ5-desaturases, respectively)were acting on acyl-CoA substrates. It is reasonably expected that theactivities of these genes will increase in subsequent generations oftransgenic plants when homozygosity is reached, and that levels of theLC-PUFA products will increase as a consequence.

The presence of the silencing suppressor in the constructs pJP3057 andpJP3059 increased both the total level of the new fatty acids in theseedoil, and the level of the final product of the pathway, DHA.

Discussion

With regard to Examples 13 to 15, introduced exogenous nucleic acids canbe detected by plants as foreign DNA or RNA leading to reducedexpression due to host-mediated transgene suppression mechanisms. Thesesuppression mechanisms may target transgenes via the biogenesis of smallRNA populations, and these small RNAs guide the suppression apparatus tolimit expression of the transgene (Matzke et al., 2001). Transgeneexpression can be limited in various ways including direct modificationsof the DNA at the site of insertion in the chromosome, such as bymethylation, or by post-transcriptional silencing at the RNA (Hamiltonand Baulcombe, 1999; Voinnet et al., 2003) or protein (Brodersen et al.,2008) level. The features of foreign DNA or RNA that trigger suchsuppression mechanism are not well understood (Lindbo et al., 1993;Lechtenberg et al., 2003). However, such host-mediated suppression oftransgene expression is more likely for traits that require highexpression, multiple transgenes and transgenes with regions ofsimilarity with each other or to the host genome (Schubert et al.,2004). Furthermore transgene performance can progressively degrade witheach subsequent generation, most likely due to DNA-methylation ofpromoter and coding regions of transgenes (Hagan et al., 2003).

Here we demonstrate that viral suppressor proteins (VSP) expressed frompost-embryogenesis seed-specific promoters are developmentally toleratedin Arabidopsis. Co-expression of various VSP with a quantifiable trait,GFP, indicated that recombinant traits were also tolerated in VSPexpressing seed (Example 14). As VSP are known to block small RNAmetabolism that constitute the transgene suppression apparatus, wesuggest that the co-expression of a VSP with recombinant traits in seedswill ensure that these traits perform at a high and undiminished levelover many generations.

As the plants tolerated VSP expression such as P19 and PePo afterembryogenesis, this suggested that endogenous developmental signals, atleast those using small RNAs, are minor or less critical at this latestage of plant development. The four VSPs chosen for this study arelikely to act upon different parts of the small RNA biogenesis andtherefore function to different extents. By reducing the silencingeffect in multi-gene transgenic cassettes via the use of a co-introducedVSP, a number of changes on transgenic expression strategies can beenvisioned. Firstly, the same expression cassettes can be usedrepeatedly with less requirement to avoid sequence repetition betweenregulatory sequences or coding regions. This feature therefore can allowlarge multi-gene expression vectors to be built using the samepromoter-polyadenylation signals. Alternatively, multiple copies of asingle gene can be used to increase expression levels with reducedlikelihood or extent of silencing effects occurring, or with increasedstability of expression over plant generations.

Example 16 Transient Expression of Genes in Plant Leaf Cells UsingSeed-specific Promoters

The enzyme activities of the proteins encoded by the Micromonas CCMP1545Δ6-desaturase (SEQ ID NO:8 encoded by SEQ ID NO:7), Pyramimonas CS-0140Δ6-elongase (SEQ ID NO:4 encoded by SEQ ID NO:3), Pavlova salinaΔ5-desaturase (SEQ ID NO:26 encoded by SEQ ID NO:25), PyramimonasCS-0140 Δ5-elongase (SEQ ID NO:6 encoded by SEQ ID NO:5) and Pavlovasalina Δ4-desaturase (SEQ ID NO:73 encoded by SEQ ID NO:72) genes, eachunder the control of seed-specific promoters, were demonstrated in leaftissue, in planta, using an enhanced Nicotiana benthamiana transientexpression system, as follows.

The chimeric vector pJP3057 described in Example 15 and containing fiveDHA biosynthesis genes, each under the control of the seed-specifictruncated napin promoter, FP1, was introduced into Agrobacteriumtumefaciens strain AGL1. A chimeric vector, designated 35S:LEC2, wasgenerated by cloning a codon-optimised Arabidopsis thaliana LEAFYCOTYLEDON2 (Arath-LEC2) gene into the EcoRI site of 35S:pORE04. The35S:LEC2 construct was introduced separately into Agrobacteriumtumefaciens strain AGL1. Transgenic cells from separate cultures of AGL1containing either pJP3057 or 35S:LEC2 were mixed and the mixtureinfiltrated into leaf tissue of Nicotiana benthamiana plants. The plantswere grown for a further four days after infiltration before leaf discswere taken for GC analysis of the total fatty acids in the leaf lipid,and of separated lipid fractions. This revealed that these genes werefunctioning to produce DHA in Nicotiana benthamiana (Table 19). Leaftissue transformed with these genes contained SDA (1.2%), ETA (2.0%),EPA (0.6%), DPA (1.7%) and DHA (2.5%). The leaf tissue also containedGLA (2.4%) and trace levels of other long-chain ω6 fatty acids.

The chimeric vectors pJP3115 and pJP3116 (Example 17) were introducedinto Agrobacterium tumefaciens strain AGL1. Transgenic cells from fourseparate cultures of AGL1 containing one of pJP3115, pJP3116, 35S:P19and 35S:LEC2 were mixed and the mixture infiltrated into leaf tissue ofNicotiana benthamiana plants. The plants were grown for a further fourdays after infiltration before leaf discs were taken for GC analysiswhich revealed that these genes were functioning to produce DHA inNicotiana benthamiana (Table 19). Leaf tissue transformed with thesegenes contained SDA (5.6%), ETA (1.4%), EPA (0.2%), DPA (1.7%) and DHA(2.4%). The leaf tissue also contained trace levels of long-chain ω6fatty acids.

This experiment confirmed that the dual constructs pJP3115 and pJP3116were functioning in combination to produce DHA as efficiently as asingle construct containing all eight genes.

TABLE 19 Fatty acid composition (percent of total fatty acids) ofNicotiana benthamiana leaf tissue transiently transformed with variousconstructs. Errors denote standard deviation between separateinfiltrations performed in triplicate. pJP3057 + P19 only 35S:LEC2pJP3057 35S:LEC2 Usual FA 16:0 13.2 ± 0.5  12.8 ± 1.0  13.3 ± 0.1  13.2± 0.6  16:1^(Δ3t) 1.5 ± 0.1 1.4 ± 0.2 1.3 ± 0.1 1.1 ± 0.016:3^(Δ 9,12,15) 7.6 ± 0.3 8.2 ± 0.4 7.1 ± 0.3 7.5 ± 0.4 18:0 1.7 ± 0.22.0 ± 0.4 1.8 ± 0.1 2.4 ± 0.3 18:1^(Δ 9) 0.9 ± 0.1 0.9 ± 0.1 1.1 ± 0.11.5 ± 0.2 18:2^(Δ 9,12) 12.6 ± 1.1  12.8 ± 0.6  13.8 ± 0.1  12.7 ± 0.4 18:3^(Δ 9,12,15) 58.3 ± 1.9  56.3 ± 2.6  56.3 ± 0.7  44.8 ± 2.1  20:00.3 ± 0.0 0.4 ± 0.1 0.3 ± 0.0 0.5 ± 0.1 Other minor 3.8 4.6 3.9 4.8Total 99.9  99.6  98.9  88.5  New Δ6 PUFA 18:3^(Δ 6,9,12) — 0.1 ± 0.0 —2.4 ± 0.1 20:3^(Δ 8,11,14) 0.1 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 0.2 ± 0.120:4^(Δ 5,8,11,14) — — — — 22:4^(Δ7,10,13,16) — — — 0.6 ± 0.122:5^(Δ 4,7,10,13,16) — — — 0.3 ± 0.0 Total 0.1 0.4 0.2 3.5 New Δ 3 PUFA18:4^(Δ 6,9,12,15) — — 0.9 ± 0.1 1.2 ± 0.1 20:4^(Δ 8,11,14,17) — — — 2.0± 0.1 20:5^(Δ 5,8,11,14,17) — — — 0.6 ± 0.0 22:5^(Δ 7,10,13,16,19) — — —1.7 ± 0.1 22:6^(Δ 4,7,10,13,16,19) — — — 2.5 ± 0.2 Total — — 0.9 8.0Total new FA 0.1 0.4 1.1 11.5  Total FA 100.0  100.0  100.0  100.0 Discussion: Rapid Failure and Validation of Seed-specific Constructs

The experiments using a transcription factor, in this case LEC2, incombination with a suite of genes each under the control of atissue-specific promoter such as a seed-specific promoter showed thatsuch constructs can be tested in a heterologous system, such as leaves,where the tissue-specific promoter would not normally be expressed, andare predictive of expression in the seed. The ability to transientlyexpress a seed-specific promoter in a leaf cell allows for rapidvalidation of construct design. Experiments to determine theeffectiveness of seed-specific promoters, especially in a multi-geneconstruct context, previously relied on stable transformation into anoilseed model plant or crop, followed by the generation of progeny linesbefore phenotypic analysis could determine the effectiveness of theconstruct in the plant seed. The fact that the levels of fatty acidsobtained in N. benthamiana were similar to those seen in stableArabidopsis transformation with this same construct as described inExample 15 increases confidence in the applicability of this assay.

Example 17 Dual-constructs for DHA Biosynthesis

The vector pJP3115 (FIG. 22) was constructed as follows. First, theSbfI-ApaI fragment of vector pJP101acq (FIG. 14) was cloned into thePstI-ApaI site of pORE03 to yield pJP3011. Next, a SwaI fragmentcontaining the codon-optimised Micromonas pusilla Δ6-desaturase (SEQ IDNO:125) was cloned into a T4 DNA polymerase-treated XhoI site in pJP3011to yield pJP3108. A SwaI fragment containing the codon-optimised Pavlovasalina Δ5-desaturase (SEQ ID NO:127) was then cloned into a T4 DNApolymerase-treated NotI site in pJP3108 to yield pJP3109. A SwaIfragment containing the codon-optimised Pyramimonas cordata Δ6-elongase(SEQ ID NO:126) was cloned into the SmaI site in pJP3109 to yieldpJP3110. The construct was then converted from a BASTA-resistantconstruct into a kanamycin-resistant construct by cloning theBsiWI-AsiSI fragment of pJP3110 into the BsiWI-AsiSI site of pORE04,yielding pJP3111. An NcoI (T4 DNA polymerase-treated)-SbfI fragmentcontaining the truncated napin promoter FP1 and the Crepis palestinaΔ12-desaturase was cloned into the EcoRV-PstI site in pJP3111 to yieldpJP3115.

The vector pJP3116 (FIG. 23) was constructed as follows. First, a SwaIfragment containing the codon-optimised Pyramimonas cordata Δ5-elongase(SEQ ID NO:128) was cloned into a T4 DNA polymerase-treated XhoI site inpJP3011 to yield pJP3112. A SwaI fragment containing the codon-optimisedPavlova salina Δ4-desaturase (SEQ ID NO:129) was cloned into the SmaIsite in pJP3112 to yield pJP3113. A NotI fragment containing the Perillafrutescens Δ15-desaturase was then cloned into the NotI site in pJP3113to yield pJP3114. The construct was then converted from aBASTA-resistant construct into a hygromycin-resistant construct bycloning a XbaI-MluI fragment containing an hygromycin resistancecassette (consisting of the Cauliflower mosaic virus 35S promoterfollowed by the CAT-1 intron-interrupted hygromycin B phosphotransferasegene obtained from the binary vector pWVEC8 and the NOS terminator) intothe AvrII-MluI site of pJP3114 to yield pJP3116.

The chimeric vectors pJP3115 and pJP3116 were introduced individuallyinto Agrobacterium tumefaciens strain AGL1 and transgenic cells fromcultures of these were mixed with AGL1 transformed with 35S:P19 and themixture infiltrated into leaf tissue of Nicotiana benthamiana plants inthe greenhouse. The plants were grown for a further five days afterinfiltration before leaf discs were taken for GC analysis which revealedthat these genes were functioning to produce DHA in Nicotianabenthamiana (Table 20). Leaf tissue transformed with these genescontained SDA (5.6%), ETA (1.4%), EPA (0.2%), DPA (1.7%) and DHA (2.4%).The leaf tissue also contained trace levels of GLA, ETA and ARA. Theconversion efficiencies were as follows: 98.9% of the oleic acid in thecell was Δ12-desaturated (not significantly different from the controlsample); 95.4% of the LA in the cell was Δ15-desaturated; 18.1% of theALA produced in the cell was Δ6-desaturated; 50.4% of the SDA producedin the cell was Δ6-elongated; 75.4% of the ETA in the cell wasΔ5-desaturated; 95.4% of the EPA in the cell was Δ5-elongated; 58.5% ofthe DPA in the cell was Δ4-desaturated to produce DHA.

Both constructs were used to transform canola. T1 seeds will becollected and analysed for general morphological changes and levels ofLC-PUFA synthesis.

TABLE 20 Fatty acid composition (percent of total fatty acids) ofNicotiana benthamiana leaf tissue transiently transformed with variousconstructs. Errors denote standard deviation between separateinfiltrations performed in triplicate. pJP3115 + pJP3116 + P19 only35S:LEC2 35S:LEC2 Usual FA 16:0 13.2 ± 0.5  12.8 ± 1.0  16.1 ± 0.1 16:1^(Δ3t) 1.5 ± 0.1 1.4 ± 0.2 1.3 ± 0.1 16:3^(Δ9,12,15) 7.6 ± 0.3 8.2 ±0.4 6.8 ± 0.1 18:0 1.7 ± 0.2 2.0 ± 0.4 3.4 ± 0.0 18:1^(Δ9) 0.9 ± 0.1 0.9± 0.1 0.7 ± 0.0 18:2^(Δ9,12) 12.6 ± 1.1  12.8 ± 0.6  2.6 ± 0.118:3^(Δ9,12,15) 58.3 ± 1.9  56.3 ± 2.6  51.0 ± 0.1  20:0 0.3 ± 0.0 0.4 ±0.1 0.6 ± 0.0 Other minor 3.8 4.6  5.7 Total 99.9  99.6  88.3 New Δ6PUFA 18:3^(Δ6,9,12) — 0.1 ± 0.0 0.2 ± 0.0 20:3^(Δ8,11,14) 0.1 ± 0.1 0.3± 0.1 0.1 ± 0.1 20:4^(Δ5,8,11,14) — — — 22:4^(Δ7,10,13,16) — — —22:5^(Δ4,7,10,13,16) — — 0.1 ± 0.0 Total 0.1 0.4  0.4 New Δ3 PUFA18:4^(Δ6,9,12,15) — — 5.6 ± 0.1 20:4^(Δ8,11,14,17) — — 1.4 ± 0.120:5^(Δ5,8,11,14,17) — — 0.2 ± 0.0 22:5^(Δ7,10,13,16,19) — — 1.7 ± 0.022:6^(Δ4,7,10,13,16,19) — — 2.4 ± 0.1 Total — — 11.3 Total new FA 0.10.4 11.7 Total FA 100.0  100.0  100.0 Discussion

pJP3115 and pJP3116 were designed to provide, in combination, all of thegenes for production of DHA, namely the two recombinant vectorscomplement each other to constitute the pathway. The fatty acid producedby the Δ12-desaturase encoded by pJP3115 was used as substrate by theΔ15-desaturase encoded by pJP3116 which also contained genes for thesubsequent Δ6-desaturase, Δ6-elongase and Δ5-desaturase. The product ofthe Δ5-desaturase, EPA, was then acted on by the Δ5-elongase encoded bypJP3115, the product of which was converted to DHA by the Δ4-desaturasealso encoded by pJP3115. The principle of dividing the transgenesbetween two constructs, which were used to separately stably transformplants with subsequent crossing of elite plants to constitute the entirepathway, avoided some of the problems associated with containingnumerous transgenes in a single construct, such as reducedtransformation efficiency due to increased size and reduced geneexpression. The combination of stable transformations of theseconstructs, either by super-transformation or by crossing two transgeniclines, will result in a transgenic plant containing the full complementof genes required for DHA synthesis.

It was also noted that construct pJP3115 contained four genes expressedin an inverted format i.e. two genes in one orientation and two genes inthe other, so that the pairs of genes were transcribed in a divergentfashion (away from each other). When compared to the inverted designused to express three genes in construct pJP107 (Example 8), it wasconcluded that the addition of a fourth gene in this format did nothinder the expression of the genes.

It was interesting to note the relatively low Δ6-elongation efficiency(50.4%) compared to other experiments described above, which was likelydue to the fact that the genes encoding the enzymes for the previousthree desaturation steps were all expressed from the FP1 promoterwhereas the gene encoding the Δ6-elongase was driven by the Arabidopsisthaliana FAE1 promoter. This was thought to cause a difference inpromoter timing, with the FAE1 promoter being activated after the FP1promoter. Compared to previous experiments where the Δ6-elongase wasdriven by the FP1 promoter, this resulted in a higher accumulation ofSDA which was then removed from the metabolic pool accessed by theΔ6-elongase before it could be acted on by the Δ6-elongase.

Example 18 Isolation and Characterisation of a Gene Encoding aMicroalgal DGAT2

Synthesis of a Full Length Micromonas pusilla DGAT2 Gene

The Micromonas CCMP1545 filtered protein models genome sequence producedby the US Department of Energy Joint Genome Institute(http://www.jdi.doe.gov/) was analysed with the BLASTP program using aputative amino acid sequence from Ostreococcus lucimarinus, GenbankAccession No. XP_00141576, as the query sequence. This analysis revealedthe presence of a predicted protein in Micromonas CCMP1545 that hadhomology with XP_00141576. The Micromonas CCMP1545 predicted proteinsequence was used to design and synthesize a codon-optimized nucleotidesequence that was most suitable for expression in dicotyledonous plantssuch as Brassica napus. The nucleotide sequence of the protein codingregion is given in SEQ ID NO:107. The plasmid construct was designated0928814 Mic1545-DGAT2 pMA. The amino acid sequence is shown as SEQ IDNO:108. BLASTP analysis using the Micromonas CCMP1545 desaturase aminoacid sequence SEQ ID NO:108 as query to other proteins in the Genbankdatabase showed that the protein had homology with DGATs. The highestdegree of identity was 53% along the full-length with the amino acidsequence of Accession No. XP_002503155, the sequence of a MicromonasRCC299 putative protein. This gene contains a diacylglycerolacyltransferase motif (NCBI conserved domain pfam03982) at amino acids74 to 334.

A genetic construct 35S:Mic1545-DGAT2 encoding the DGAT2 under thecontrol of the constitutive 35S promoter was made by inserting theentire coding region of 0928814_Mic1545-DGAT2_pMA, contained within anEcoRI fragment, into 35S-pORE04 (Example 4, above) at the EcoRI site,generating pJP3128. This chimeric vector was introduced intoAgrobacterium tumefaciens strain AGL1 and transgenic cells from culturesof these were mixed with 35S:P19 AGL1 and the mixture infiltrated intoleaf tissue of Nicotiana benthamiana plants in the greenhouse. Theplants were grown for a further five days after infiltration before leafdiscs were taken for lipid analysis which revealed that the geneencoding DGAT2 was functioning to increase total TAG in transformed leafcells, with preference for polyunsaturated fatty acids (Table 21). Inparticular, the level of polyunsaturated fatty acids in TAG in thetransformed cells increased at least 3-fold.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from U.S. 61/199,669 filed 18Nov. 2008, and U.S. 61/270,710 filed 9 Jul. 2009, the contents of bothof which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

TABLE 21 Effect of expression of the Micromonas pusilla DGAT2 inNicotiana benthamiana leaf. P19 is the control, DGAT2 also contains P19.The total amount of TAG in the leaf tissue increases two-fold when theDGAT2 is expression and polyunsaturated fatty acids are favoured. AmountAmount in TAG, ug TAG profile in PL, ug PL profile P19 DGAT2 P19 DGAT2P19 DGAT2 P19 DGAT2 C16:0 0.73 0.86 55.8 32.7 C16:0 26.33 20.37 14.214.5 C16:1d7 0.00 0.00 0.0 0.0 C16:1d7 0.00 0.00 0.0 0.0 16:1d13t 0.000.00 0.0 0.0 16:1d13t 3.40 2.96 1.8 2.1 16:2w6 0.00 0.00 0.0 0.0 16:2w61.60 1.19 0.9 0.8 16:2w4 0.00 0.05 0.0 1.7 16:2w4 1.15 1.00 0.6 0.716:3w3 0.00 0.00 0.0 0.0 16:3w3 12.65 9.05 6.8 6.4 C18:0 0.27 0.32 20.712.0 C18:0 4.50 3.56 2.4 2.5 C18:1d9 0.00 0.21 0.0 8.0 C18:1d9 1.89 1.821.0 1.3 C18:1d11 0.00 0.00 0.0 0.0 C18:1d11 0.85 0.78 0.5 0.6 C18:2n60.14 0.68 11.0 25.9 C18:2n6 24.36 16.52 13.2 11.7 C18:3n6 0.00 0.00 0.00.0 C18:3n6 0.00 0.00 0.0 0.0 C18:3n3 0.16 0.52 12.5 19.6 C18:3n3 106.3882.16 57.6 58.4 C20:0 0.00 0.00 0.0 0.0 C20:0 0.59 0.51 0.3 0.4 C18:4n30.00 0.00 0.0 0.0 C18:4n3 0.00 0.00 0.0 0.0 C20:3n3 0.00 0.00 0.0 0.0C20:3n3 0.34 0.00 0.2 0.0 C22:0 0.00 0.00 0.0 0.0 C22:0 0.37 0.37 0.20.3 C20:4n3 0.00 0.00 0.0 0.0 C20:4n3 0.00 0.00 0.0 0.0 C20:5n3 0.000.00 0.0 0.0 C20:5n3 0.00 0.00 0.0 0.0 C22:3n3 0.00 0.00 0.0 0.0 C22:3n30.00 0.00 0.0 0.0 C24:0 0.00 0.00 0.0 0.0 C24:0 0.43 0.41 0.2 0.3C22:5n6 0.00 0.00 0.0 0.0 C22:5n6 0.00 0.00 0.0 0.0 C22:5n3 0.00 0.000.0 0.0 C22:5n3 0.00 0.00 0.0 0.0 C22:6n3 0.00 0.00 0.0 0.0 C22:6n3 0.000.00 0.0 0.0 TFA 26.2 52.7 100.0 100.0 TFA 3696.9 2814.0 100.0 100.0(ug/g If (ug/g If FW) FW)

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The invention claimed is:
 1. A process for producing seed comprisingeicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid,the process comprising the steps of: (i) growing a Brassica napus plantcomprising exogenous polynucleotides encoding a microalgal Δ6-desaturasewhich has greater Δ6-desaturase activity on ALA-CoA as fatty acidsubstrate than on ALA joined to the sn-2 position of phosphatidylcholine(PC) as the fatty acid substrate, a Δ6-elongase, a microalgalΔ5-desaturase, a microalgal Δ5-elongase, and a microalgal Δ4-desaturase,wherein each polynucleotide is operably linked to one or more promotersthat direct expression of said polynucleotides during the development ofthe seed, to produce seed that comprises eicosapentaenoic acid,docosapentaenoic acid and docosahexaenoic acid which were produced fromα-linolenic acid (ALA) during development of the seed and which arepresent in an esterified form as part of triglycerides at levels basedon an efficiency of conversion of α-linolenic acid to eicosapentaenoicacid, docosapentaenoic acid or docosahexaenoic acid of at least 17.3%and an efficiency of conversion of eicosapentaenoic acid todocosapentaenoic acid of at least 60%, and (ii) harvesting the seed fromthe plant.
 2. The process of claim 1, wherein docosapentaenoic acid anddocosahexaenoic acid are present at levels based on an efficiency ofconversion of α-linolenic acid to docosapentaenoic acid ordocosahexaenoic acid of at least 15.4%.
 3. The process of claim 1,wherein docosahexaenoic acid is present at a level based on anefficiency of conversion of α-linolenic acid to docosahexaenoic acid ofat least 9.5%.
 4. The process of claim 1, wherein docosahexaenoic acidis present at a level based on an efficiency of conversion ofeicosapentaenoic acid to docosahexaenoic acid of at least 45%.
 5. Theprocess of claim 1, wherein arachidonic acid, eicosapentaenoic acid,docosapentaenoic acid and docosahexaenoic acid together comprise 15% ofthe total fatty acids in the seed.
 6. The process of claim 1, whereinthe Δ6-desaturase is an Ostreococcus tauri or Micromonas pusillaΔ6-desaturase.